CN1764671A - Light induced immobilisation - Google Patents

Abstract

The present invention involves a method of coupling disulfide bridge containing proteins to a carrier by inducing the formation of thiol groups on a protein with irradiation, and coupling the protein to the carrier. The formation of thiol group(s) in the protein is a consequence of the disruption of disulfide bridges following electronic excitation of aromatic amino acid residues located in close spacial proximity to the disulfide bridge. The light-induced coupling method facilitates the orientated immobilization of a protein on a carrier.

Description

Photoinduced fixing

Invention field

The present invention relates to make protein-crosslinking or be fixed to method on the carrier.

Background of invention

Molecule can pass through hydrophobicity or ionic interaction passively, or covalently is fixed on carrier or the solid surface by being attached to the activatory surface group.At the requirement of the huge importance of fixed that is used for the screening of solid state chemistry and biology, studied this analysis of technology purposes widely.This technology is in many different field of biotechnology, is widely used in molecule fixing in for example diagnosis, biosensor, affinity chromatography and the elisa assay.The recent development of dna microarray has confirmed the value of technique for fixing, in dna microarray multiple oligonucleotide or cDNA sample is fixed on the solid surface in the addressable mode in space.By helping the genetic expression in the holistic approach living organism, these arrays have brought reform completely for genetics research.Develop similar method and be used for analysis of protein, wherein only needed few protein binding to 1 pik each to the microarray to put the analysis that is used for subsequently.Can analyze the proteic function or the structural performance that are attached to this microarray then, thereby be convenient to screen with the scale and the speed of previous the unknown.Can use the biomolecules that is bonded to solid surface to catch to be present in other binding molecule not in the mixture in addition.

In recent years, for biomolecules being fixed on the solid surface in the mode that control is arranged, make bound fraction that the least surface migration is arranged simultaneously, and the purpose that keeps its natural structure and function fully, the exploitation of this technology has become the theme (Veilleux J (1996) IVD Technology, March p.26-31) of further investigation.The proteopexy of simple types has utilized surface and the high inherent binding affinity of common albumen.For example albumen comprises that by means of many weak contacts Van der Waals interacts and hydrogen bonding interacts and physically be adsorbed onto hydrophobic substrate.The advantage of this method is that it is avoided treating bonded albumen and modifies.On the other hand, so bonded albumen may be distributed in solid support unevenly and/or may be inactivation, because for example it clusters and may cause the steric hindrance of avtive spot/land in any functional examination subsequently.

The fixed alternative approach depends on utilizes some strong covalent bonds to make protein binding to solid surface (Wilson D.S., Nock S., 2001, Current Opinion in ChemicalBiology 6:81-85).Example comprises makes biotinylated proteopexy to the upholder that scribbles streptavidin, and the proteopexy that makes the His-mark that contains the polyhistidyl sequence is to chelating Ni ²⁺Upholder on.Can be used for being attached to other functional group on the protein surface on suitable surface comprise by means of Schiff's base make amine and aldehyde reaction, make amido and have the amine of glutaraldehyde surface-crosslinked with form peptide bond, make the hydroxy-acid group that is present on albumen and the support surface with carbodiimide is crosslinked, based at formation disulfide linkage between 2 thiol groups with between thiol group and gold surface, form the crosslinked of mercaptan-Au key.

The amine coupling is widely used fixedly chemical process.The N-hydroxy-succinamide ester by the carboxy moiety of Sensor Chip CM 5 matrix through reacting in water with N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylamino-propyl) carbodiimide hydrochloride (EDC) and forming, its then spontaneously with albumen on amido reaction to form covalent linkage (Johnsson B., Deng the people, 1991, Anal Biochem 198:268-77).After fixing, make with 1M thanomin hydrochloride that unreacted N-hydroxy-succinamide ester inactivation does not have protein-bonded zone with sealing on the upholder.This method is heavy, and this is because of employed reagent in each step that must remove before beginning next step usually in the chemical fixation method.

Veilleux J (1996) (seeing above) has described through the disulfide linkage method for immobilizing biomolecules.Use weak reductant, for example dithiothreitol (DTT), 2 mercapto ethanol or three (2-propyloic) phosphine hydrochloride are handled protein sample with the disulfide linkage between the reduction cysteine residues, and it is bonded to the support surface that is coated with maleimide then.After this primary amine group on alternatively available 2 Iminothiolane hydrochloride (TrautShi reagent) modified protein secures it to the maleimide surface to import new sulfydryl.Demonstration through disulfide linkage with proteopexy on golden substrate, can be used for from the Cupredoxin aleuroplast of willow blue plain (Andolfi, people such as L. 2002, Arch.Biochem.Biophys.399:81-88).Because this hypoproteinosis disulfide linkage, so replace residue Ile21 and the Glu25 that the surface exposes simultaneously with Cys.Confirmed between the halfcystine that inserts, to form disulfide linkage from the 3D crystalline structure of the sudden change plastocyanin of purifying.The sudden change plastocyanin of expressing in the born of the same parents in bacterium is exposed to reducing environment in tenuigenin, thereby the halfcystine that make to insert is reduced, and therefore can mediate isolating albumen and directly be adsorbed onto on the golden substrate.Therefore proteic thiol group binding characteristic depends on the interior or external chemical reduction of body of cysteine residues on the protein surface.

Described be used for rnase (RNA enzyme), make thiol group binding characteristic through engineering approaches enter alternative approach in the albumen, this rnase has 4 essential Gelucystines (Sweeney, people such as R.Y. 2000 Anal Biochem.286:312-314).Single in this case cysteine residues has replaced Ala19, and it is arranged near the surface ring of N-end of RNA enzyme A.Halfcystine in the RNA enzyme of expressing is as being protected with the mixed disulfide of 2-nitro-5-thiobenzoic acid.After going protection with excessive dithiothreitol (DTT) subsequently, the RNA enzyme is not lost enzymic activity with being attached to the iodoacetyl coupling of crosslinked agarose resin.In addition, it is exposed to the protein requirement that preparation is used for fixing protective material and goes in the protective material, and this may its natural structure of negative impact and/or function.

US 6,350, described the enzyme that is used for relying on FAD in 368 and be coupled to method on the electrode, be bonded to can chemical association (for example passing through thiol group) or to be attached to the functionalization FAD of the lip-deep bound fraction of electrode (for example golden) compound for pheron and covalent linkage thus.Further make fixed FAD-enzyme complex functionalization and can use it for the electro-chemical systems that makes analyte or optical signal distortion with the electron mediator group.

Also after deliberation photoinduced technique for fixing, cause with naphtoquinone compounds be used for photochemistry be connected to the bag carbonaceous upholder (EP0820483).With activating behind the non-ionized electromagnetic radiation irradiation of UV-light to the visible-range.Some zone that can use hovel to activate upholder is adhered to the biomolecules that is used for subsequently.Behind the compound of irradiates light chemical activation, anthraquinone will react and form stable ehter bond with polymer surfaces as free radical.Owing in natural biomolecules, do not find anthraquinone, suitable part must be imported biomolecules.With regard to albumen, additional sample preparation steps may need thermochemistry to be coupled to quinone and may not be site-specific.

At US 5,412,087 and US 6406844 in further developing of photoinduced technique for fixing disclosed, the method that is used to prepare the joint that is bonded to substrate has wherein been described.Provide for the end of linkers and use up removable protecting group, for example the reactive functional groups of nitro-aromatics protection.After the exposure, protecting group is lost, and joint can be at its amino or C-terminal and monomer, and for example amino acid reacts.The similar removable protecting group of light of the own portability of this monomer in addition, it also can be substituted by light during the subsequent reaction cycle.This method has specific solid phase synthesis to be used, but being not easy to albumen combines with the orientation of upholder.

At US 3,959, disclose in 078 be used for fixing enzyme have thermochemistry and the functional substituent difunctionality reagent of photochemistry.The derivative of aromatic yl azide (arylazide) has been described, its make azido-can with enzyme and the activation and the covalent coupling that carry out the light mediation with the substituting group of solid support thermal chemical reaction.Can not control the direction of institute's fixed enzyme molecule by this method.

In people (1993) Bioconjugate Chem.4:528-536 such as WO 91 16425 and Collioud A, described between the wetting agent N-[that utilizes Heterobifunctional-[3-(trifluoromethyl) diazacyclo propane-3-yl] phenyl]-4-maleimide butyramide (N-[m-[3-(trifluoromethyl) diazirin-3-yl] phenyl]-4-maleimidobutyramine) with albumen directionally, light dependency ground, the method for Covalent Immobilization on solid support.Aryl diazacyclo propane (aryldiazirine) sense of this cross-linking reagent can promote that light is dependent, the Cabbeen mediation, covalent bonding is to inert solid support or biomolecules, for example albumen, carbohydrate and nucleic acid.The maleimide official of linking agent can make it possible to be bonded to the mercaptan surface by the thermochemistry modification of halfcystine mercaptan.Can interact by the thermochemistry between the thiol group that exposes on maleimide official energy and the protein surface and obtain cross-linking reagent and proteic directed the combination, yet this processing may be modified the structure and the activity of target protein.Yet cross-linking reagent can be that it can not provide target protein that the direction of control is arranged with the shortcoming of proteic photoinduction covalent coupling through the Cabbeen official.

For the fixing means that great majority are described, its common ground is to use one or more thermochemistry/chemical steps, uses hazardous chemicals sometimes, and some of them have deleterious effect to protein-bonded structure and/or function probably.Existing method often is invasive, thus external group is imported albumen to play functional group, and this can cause protein denaturation, and reduces its biological activity and substrate specificity.

In albumen coupling and fixed field, need to improve the link coupled method, wherein kept the 26S Proteasome Structure and Function characteristic and the may command link coupled direction of coupling or frozen composition.

Summary of the invention

The invention provides albumen or peptide are coupled on the carrier through stable key (covalent linkage or mercaptan-Au key), keep the natural structure and the functional performance of coupling protein or peptide simultaneously, and avoid using the method for the step of one or more chemical.This forms contrast with being used for protecpectic traditional coupling method, and traditional method generally comprises several chemical reactions, and it may be expensive, time-consuming and harmful to protein-bonded structure/function.The direction of may command the method according to this invention institute link coupled albumen or peptide in addition, so that preserve its characteristic, enzyme characteristic for example.By comparison, most of known protein coupling method causes albumen to be fixed on the carrier with at random direction, and it is attended by the remarkable risk that reduces biological activity and increase limit of detection.The present invention utilizes the inherent nature of albumen and peptide, and wherein after using the light Long-Duration Exposure that can be absorbed by aromatic amino acid, the disulfide linkage of contiguous aromatic amino acid residue destroys in albumen or the peptide.Use the thiol group that produces by photoinduced disulfide linkage destruction in albumen or the peptide that albumen or peptide are fixed to carrier then.

According to a first aspect of the invention, provide to make the albumen that comprises disulfide linkage or peptide be coupled at method on the carrier, comprise the following steps: irradiation albumen or peptide and produce thiol group, and with the albumen of irradiation or peptide and carrier incubation that can the combined sulfur alcohol radical.In addition, by making albumen or peptide with carrier incubation that can the combined sulfur alcohol radical, with irradiation albumen or peptide when carrier exists in albumen or peptide, to produce thiol group, obtain thus with carrier on the coupling of mercaptan part conjugated group, and make coupling of the present invention become possibility.According to this coupling or fixed method, irradiation comprises the light of the wavelength that excites one or more aromatic amino acids.The aromatic amino acid of irradiation can comprise tryptophane, tyrosine and phenylalanine in the albumen, or alternatively aromatic amino acid is a tryptophane.The further aspect according to the present invention, irradiation are included near the wavelength 295nm, 254nm or the 275nm.The further aspect according to the present invention, the albumen of irradiation or peptide are coupled on the carrier that comprises peptide or albumen or any other biomolecules.Alternatively, the albumen of irradiation or peptide and comprise the upholder of gold or with the upholder of mercaptan conjugated group derivatize or comprise thiol group or the upholder coupling of disulfide linkage, wherein the coupling with upholder may be a kind of fixing.Randomly carrier or upholder comprise transcribed spacer.The further aspect according to the present invention provides to comprise according to making albumen or peptide be coupled at the coupling protein that open method produced on the carrier or the carrier or the upholder of peptide.The further aspect according to the present invention comprises enzyme, transcription factor, protein structure domain, antigen, antibody or conjugated protein and other albumen or peptide according to making albumen or peptide be coupled at open method link coupled albumen on the carrier or peptide.The further aspect according to the present invention comprises medicine on the pharmacopedics according to making albumen or peptide be coupled at open method link coupled carrier on the carrier.Further aspect is to comprise according to the carrier or the upholder that make albumen or peptide be coupled at the coupling protein that open method produced on the carrier being used for the purposes that the test kit of biological functionality mensuration was measured or be used to provide to biological functionality according to the present invention.Thisly be used for the purposes that biological functionality measures and comprise: biosensor, chromatography, immunodetection, enzymatic determination, Nucleotide are in conjunction with detection, protein-protein interaction, protein modified, carrier target or targeting proteins.In aspect the present invention is further, be provided for identifying the method that can comprise the albumen or the peptide of disulfide linkage by the irradiation destructive.

The accompanying drawing summary

Fig. 1 shows the fluorescent emission intensity as the 350nm place at of the function of 295nm place irradiation time.

Fig. 2 shows proteic SH group and 5, and 5 '-dithio is two-and linked reaction between (2-nitrobenzoic acid) SS group (DTNB) and the stoichiometry of nitro thiobenzoate (NTB) discharge.

Fig. 3 shows as the at of the function of 295nm place irradiation time in the new concentration (hollow circle) of the free sulphur alcohol radical of formation of the increase [F/Fo] (solid line) of the Trp at 350nm place fluorescence (F) emissive porwer and the special ratio [F/Fo] that increases with fluorescent emission in the at sample.

Fig. 4 is presented at (A) does not have the DTT[solid circles in 25 ℃]; (B) there is not the solid black line of DTT[in 70 ℃]; (C) be heated to 70 ℃ and be cooled to 25 ℃ after, do not have the hollow circle of DTT[in 25 ℃]; (D) be heated to 70 ℃, add DTT and also be cooled to 25 ℃, have the solid gray line of DTT[in 25 ℃] incubation period between after with the 295nm optical excitation, the fluorescence emission spectrum of 1 μ M at solution.

Fig. 5 is presented at the spectral response curve of aromatic amino acid residue in the aqueous solution of pH7.0.

Fig. 6 shows the structure of the probe Bodipy FL-L-Gelucystine of intramolecularly quencher, and its SS key destroys when having free SH group, and the result forms the fluorescence of Bodipy group.

Fig. 7 show by with the reaction of Bodipy FL-L-Gelucystine, before 296nm place UV-irradiation and afterwards, the detection of the SH group that dissociates in the protein sample.Shown (A) through the sudden change at (W69A) of following processing; The Bodipy group fluorescence emission spectrum of natural at (B) protein sample: do not shine and deduct Bodipy probe (); Do not shine and have Bodipy probe (●), and the processing with irradiation (zero) of bodipy.

Fig. 8 show by with the reaction of Bodipy FL-L-Gelucystine, before 296nm place UV-irradiation and afterwards, the detection of the SH group that dissociates in the protein sample.Shown through following processing hen egg-white lysozyme (lysosyme) (A); Snow-white head mold (Rhizopus niveus) TGL (B); Human plasminogen (C); Placental alkaline phosphatase (D); Rennin (E); The Bodipy group fluorescence emission spectrum of human normal immunoglobulin (F) protein sample: do not shine and deduct the Bodipy probe; Do not shine and have a Bodipy probe; And processing with irradiation of bodipy.

Fig. 9 shows near the spheric contour plot of the different spaces Trp S-S triplet in the 8 radiuses.Draw different triplets along X-axis, and according to itself and at triplet (1cex; Graduation is for No.1) the similarity graduation, and draw different residue groups along Y-axis.＞1 score value is meant that residue/group was performance (over-represented), and＜1 score value is meant that residue/group owes performance (under-represented).

Figure 10 shows near the spheric contour plot of the different spaces Trp S-S triplet in the 8 radiuses.Draw different triplets along X-axis, and according to itself and at triplet (1cex; Graduation is for No.1) the similarity graduation, and draw different residue groups along Y-axis.＞1 score value is meant that residue/group was performance, and＜1 score value is meant that residue/group owes to show.In this analysis, comprise 2 Trp S-S triplets in the alpha-lactalbumin, find that wherein Trp60-Cys73-Cys91 is destructible when ultraviolet light irradiation, and Trp118-Cys28-Cys111 is not destructible.

Figure 11 is presented near the spheric isogram of the interior different spaces Trp S-S triplet of the 8 radiuses of identifying among the IGG1 triplet A that is present in immunoglobulin (Ig) etc.Draw different triplets along X-axis, and according to itself and at triplet (1cex; Graduation is for No.1) the similarity graduation, and draw different residue groups along Y-axis.＞1 score value is meant that residue/group was performance, and＜1 score value is meant that residue/group owes to show.

Figure 12 is presented near the spheric contour plot of different spaces Trp S-S triplet in the 8 radiuses of identifying among the IGG1 triplet B that is present in immunoglobulin (Ig).Draw different triplets along X-axis, and according to itself and at triplet (1cex; Graduation is for No.1) the similarity graduation, and draw different residue groups along Y-axis.＞1 score value is meant that residue/group was performance, and＜1 score value is meant that residue/group owes to show.

Figure 13 shows, on the quartzy slide glass of SH-activatory fixedly during the at, continuously or under restriction shines of 296nm place, in the TIRF flow cell, detect excite at the 296nm place time, in the time-histories of the fluorescent emission at 330nm place.In the steps A: with the at solution flushing flow chamber of 2.0 μ M; Step B: with flow chamber and 2.0 μ M at solution incubation; Step C/D: with buffer A rinsing flow chamber; And step e: with stain remover and buffer A rinsing flow chamber.

Figure 14 A: be presented at the natural at of fixed on SH-activatory or the hydroxylated quartzy slide glass, the final fluorescent emission intensity (at the 330nm place, when exciting) under continuous or restriction irradiation at the 296nm place.B: be presented at natural at of fixed and sudden change at (W69A) on the quartzy slide glass of SH-activatory, in continuous illumination or adusk final fluorescent emission intensity (at the 330nm place, when exciting) at the 296nm place.

Figure 15 shows by the fluorescent emission intensity (at the 450nm place, when exciting at the 365nm place) of butyric acid 4-methyl umbrella shape ester (4-methylumbelliferylbutyrate) UV-irradiation, through being fixed on the at hydrolysis on SH-activatory or the hydroxylated quartzy slide glass.

Figure 16 is presented at 296nm place Continuous irradiation 10 minutes (A), or Continuous irradiation (C) and in the dark when (B), at (A) N,O-Diacetylmuramidase, (B) N,O-Diacetylmuramidase with during (C) rennin is fixed on SH-activatory or the hydroxylated quartzy slide glass, that detects in the TIRF flow cell excites at the 296nm place, in the time-histories of the fluorescent emission at 330nm place.

In Figure 16 A, B and C, in step (a/A), use albumen (N,O-Diacetylmuramidase or rennin) incubation flow chamber 10 minutes (initial flush/flowed 6.66 minutes); In step (b/B), washed flow chamber 10 minutes (initial flush/flowed 6.66 minutes) with damping fluid; In step (c/C), use Helmanex, 2% pH10-11, washing flow chamber 4-5 minute (initial flush/flowed 3.33 minutes); And in step (d/D), read (initial flow 3.33 minutes) with maintenance with damping fluid washing flow chamber.

Figure 17 A: show as the relative reactivity that shines or place the at sample (comparing) of the function under the light of laboratory at the 296nm place with the sample that places dark.B: be presented at exist 5.1mM DTT (●, ●) or the relative reactivity of at sample when not having DTT (zero).There is not the sample of DTT to measure at 1600 minutes 300 minutes data point.

Definition

As used in this, term " ultraviolet light " or " irradiation " or " UV-irradiation " or " ultraviolet light irradiation " are wave-length coverage or the single wavelength of ultraviolet light, or be used for infrared (the IR)/visible light of multiphoton excitation, it excites aromatic amino acid specifically, maybe can imitate other aromatic systems of aromatic amino acid, the wavelength of 295nm, 275nm or 254nm preferably approximately, more preferably excite the wavelength of tryptophan, tyrosine or phenylalanine, most preferably excite the wavelength 295nm of tryptophan.

As used in this term " albumen " comprise the fragment of polypeptide, albumen and antibody or any other based on amino acid whose material. In addition term " albumen " comprise enzyme, antibody, antigen, transcription factor, in conjunction with albumen, for example DBP or protein structure domain.

As used in this, term " spatial neighbor " relates to the physical distance between interior 2 chemical groups of composition, thereby three-dimensional approaching group is considered to spatial neighbor.If aromatic amino acid absorbs excitation energy behind the irradiation, the disulfide linkage of the contiguous aromatic moieties in space can play quencher in the albumen so.Spatial neighbor tryptophan residue and may play physical distance between half Gelucystine of disulfide linkage of quencher effect and can be but the scope that is not limited to 1 to 10 .

As used in this, term " carrier " can be soluble compound or polymkeric substance, or insoluble compound or polymkeric substance, wherein compound or polymkeric substance comprise the mercaptan binding partner, for example can with reactive SH of radiation-induced thiol group link coupled or SS key.The term carrier also can be another kind of biomolecules, for example another kind of albumen, peptide or polypeptide.In addition term " carrier " be understood to include comprise can with the upholder of radiation-induced thiol group link coupled material, for example golden upholder or carry the derivatize upholder of mercaptan binding partner.

As used in this, term " upholder " can be any support material, for example electronic chip, slide glass, wafer, particle, resin, hole, pipe or film, it includes but not limited to comprise any material of polymkeric substance, for example topaz, polystyrene, polyethylene, polyester, polyetherimide (polyethermides), polypropylene, polycarbonate, polysulfones, polymethylmethacrylate [PMMA], poly(vinylidene fluoride) [PVDF], siloxanes; Diamond; Glass, for example quartz and silica; Silicon, for example silicon wafer; Metal; Film, for example nylon membrane, nitrocellulose filter; Porous material, example gel, agarose or Mierocrystalline cellulose; Potteries etc., it comprises the derivative that is with or without the insertion transcribed spacer and is convenient to the upholder form of ownership of combined sulfur alcohol radical in addition.

As used in this, term " transcribed spacer " comprises a series of compound, and its objective is provides the connection between albumen and the carrier or make fixed albumen exceed the surface of upholder.

As used in this, term " medicine on the pharmacopedics " comprises the goods that are intended to be used to diagnose, cure, alleviate, treat or prevent the disease of people or other animal; With the goods (except food) of the structure or any function that are intended to influence people or other animal health, and be intended to goods as the composition of any above-mentioned specified goods.

As used in this, term " biological function mensuration " comprises that biosensor, immunodetection, enzymatic determination, Nucleotide are in conjunction with detection, chromatography, protein-protein interaction, protein modified, carrier target or targeting proteins.

As used in this, term " biosensor " comprises integration biology or biology deutero-sensor, for example Analytical equipment of amino acid (for example halfcystine), albumen, antibody, nucleic acid, microorganism or cell.Sensor is incorporated in the physical chemistry transmodulator or with the physical chemistry transmodulator and is closely related.The overall purpose of biosensor is generation and single analyte or one group of proportional discrete or successive digital electronic signal of analyte of interest.

Detailed Description Of The Invention

The present invention utilizes proteic natural characteristics, considered structural changes radiation-induced in the albumen, and described variation is considered to postpone its photodegradation.When albumen was exposed to ultraviolet light irradiation, some disulfide linkage destroyed and form activatory mercaptan.Although usually in structural core and, be the most responsive to UV light-induced destruction near the disulfide linkage of aromatic amino acid near the surface of folded protein/on the surface of folded protein, have disulfide linkage.During albumen was exposed to UV-light, the energy that is absorbed by aromatic amino acid residue side chain was transferred to contiguous disulfide linkage, and it plays quencher (Neves-Petersen MT. waits the people, 2002, ProteinScience 11:588-600).Yet, transfer to the flow of energy of disulfide linkage and the middle chemical product that may form, for example free radical/the ion that forms behind the optical excitation sample finally is used for triggering its destruction.To exist in the mode of spatial neighbor tryptophane often be naturally occurring to disulfide linkage in the albumen, show photoinduced disulfide linkage destroy be widely phenomenon (Petersen MTN. waits the people, 1999, Protein Engineering 12:535-548; People such as Neves-PetersenMT, 2002, Protein Science 11:588-600; People 2002 such as Vanhooren A, Biochemistry 10; 41 (36): 11035-11043).

The exploitation that is used for the albumen orientation is fixed on this radiation-induced phenomenon on the carrier is basis of the present invention.From the at of fungi pea fusarium solanae (Fusarium solani pisi), be to be used for photoinduced disulfide bond reduction being described and being fixed in one of several modes albumen of the method on the carrier, yet application of the present invention never only limit to pattern albumen.At is can degrade to be present in the lipolytic enzyme of the lip-deep insoluble lipid polyester matrix cutin of plant leaf.At is industrial important enzyme, and suggestion is mixed the stain remover that is used for removing fat with it.It has 2 disulfide linkage; One near avtive spot, and the far-end at proteic antipole is in close proximity to proteic single tryptophan residue.Make enzyme deactivation (Soliday CL waits the people, 1983, BiochemBiophys Res Commun 114:1017-22) near the chemical reduction meeting of the disulfide linkage of avtive spot.

In its native conformation, the single tryptophan residue of pea fusarium solanae at is owing to exist contiguous disulfide linkage but the height quencher.After the optionally irradiation at that 295nm prolongs, the fluorescence quantum yield of single Trp residue increases simultaneously along with the destruction of contiguous disulfide linkage.The quantum yield increase of Trp residue has reflected the ability of the Trp residue of the loss of disulfide linkage and quencher excited state thereof in the at.

Known disulfide linkage is the good quencher of excited state aromatic moieties.Spatially closely adjacent any aromatic moieties all can cause the photoinduced destruction of contiguous disulfide linkage.So 3 kinds of aromatic amino acids that exist in the albumen, promptly tryptophane, tyrosine and phenylalanine all are the potential media of photoinduced disulfide linkage destructive.When using when 240nm extends to the photoirradiation of 300nm wavelength region, will excite all aromatic moieties, independent aromatic moieties has different obtained the maximum absorption (table 1: the data that obtain in neutral pH).

Table 1

In water	Obtained the maximum absorption	The emission maximum value
			Phe	254nm	282nm
Tyr	275nm	303nm
			Trp	280nm	250nm

Because the excitation spectrum of aromatic amino acid residue is partly overlapping, so the albumen irradiation under single or narrow range of wavelengths is excited to different degree with independent residue.Can be used for tryptophan residue in the elicitor protein optionally at the irradiation at 295nm place.With simultaneous excitation tyrosine and tryptophan residue, it can cause photoinduced disulfide linkage to destroy simultaneously then at the irradiation at 280nm place.When carrying out irradiation, for example when carrying out two-photon excitation, with photon (light) irradiation sample with half energy (twice wavelength) of the photon that uses in single photon experiment by multiphoton excitation(MPE).For example, the UV-light at available 295nm place, or the two-photon excitation that is used in about 690nm wavelength is realized the electron excitation of tryptophane.The tyrosine residues that excites in addition can cause exciting of contiguous tryptophan residue by the mechanism that is called FRET (fluorescence resonance energy transfer), and it can cause disulfide linkage to destroy conversely.

It should be apparent to those skilled in the art that can be from proteic three-dimensional structure the position of each disulfide linkage and the contiguous amino acid prediction destruction of disulfide linkage in the given albumen under selected wavelength.Spatially the disulfide linkage with aromatic amino acid residue vicinity is least stable to UV-light probably.On inspection comprise the three-dimensional structure of the albumen subgroup of the closely adjacent space triplet TrpCys-Cys in space be positioned at the closely adjacent position of the tryptophan residue of triplet so that identify which amino acid.This is analyzed and has identified to have the triplet similar to the at albumen of contiguous amino acid composition on every side, and it can be used for predicting which albumen will have destructive triplet disulfide linkage after UV-irradiation.Can be used to predict whether it may be as follows to the amino acid component characteristic around the space TrpSS triplet in the albumen of UV light-induced destruction sensitivity: 1) the aromatic amino acid residue is positioned at 10 of disulfide linkage; 2) the emission dipole plane of expection aromatics Trp side chain can not exchange excitation energy perpendicular to those Trp-SS triplets of disulfide linkage absorption dipole planar between excited state Trp and disulfide linkage; 3) in 8 radiuses of proteic UV-light-destructible Trp S-S triplet, there is the special subgroup of amino-acid residue, as at, N,O-Diacetylmuramidase, lipase, Profibrinolysin, rennin and alpha-lactalbumin, the confirmation of (and immunoglobulin (Ig)-see below), it is different from the average frequency that amino acid exists in the general albumen.In amino acid whose these special subgroups, amidoamino acid residue (Asn, Gln) was performance, and the occurrence rate of in most of the cases short aliphatic residue (Gly, Ala, Val) and/or long aliphatic residue (Leu, Ile) also was performance.

Compare 8 dust collection of illustrative plates and 12 dusts and 15 dust collection of illustrative plates, how the isopleth that can be observed collection of illustrative plates weakens.The amino acid composition of subgroup approaches average proteic composition.It is in each triplet almost that all 3 collection of illustrative plates showing of all can making examined, and has charged amino acid group (His, Lys, Arg) (Asp, Glu) and the proline residue owing to show.

Be positioned at the amino-acid residue of Trp-SS triplet 8 radius proximities in the immunoglobulin (Ig), had the amino acid that comprises hydroxyl of performance, and aromatic moieties also be performance in many IgG Trp S-S triplets.In the amidoamino acid in many immunoglobulin (Ig)s (Asn, Gln) one or both and aliphatic residue occurrence rate also all were performances, and wherein the amino acid around the triplet of TrpSS space is formed the composition (immunoglobulin part of IGG1 triplet A group) that is very similar to around the at triplet.We also observe the charged amino acid (His, Lys, Arg) (Asp, Glu) owing to show and the group of proline residue in these immunoglobulin (Ig)s.Integrate, these parameters can be used to identify in the albumen which disulfide linkage can be coupled to photoinduction can the carrier of coupling thiol group on.

One of this albumen is the goat alpha-lactalbumin, and what wherein be positioned at amino-acid residue and at around the tryptophan residue 8 spheries of goat alpha-lactalbumin triplet is very similar.In addition, shown recently behind ultraviolet excitation goat alpha-lactalbumin that the disulfide linkage that is adjacent to tryptophan residue destroys and form free thiol group (people such as Vanhooren A, 2002, see above).Also may be fully according to its primary structure, promptly its aminoacid sequence is predicted the ultraviolet light stability of the disulfide linkage of inferring in the given albumen, prerequisite is the three-dimensional structure of known homologous protein and the homology modeling that can be used for given proteic three-dimensional structure.

The currently known methods that also it is evident that recombinant DNA technology can be used for aminoacid replacement is imported in the protein sequence, to produce the destructible disulfide linkage of additional light.This replacement can import albumen with tryptophan residue or other aromatic amino acid residue, makes the disulfide linkage of its spatial neighbor in endogenous or recombined engineeringization, or alternatively disulfide linkage can be imported in the mode of space near endogenous aromatic moieties.Alternatively aromatics and disulfide linkage can be imported in the approaching mode in space each other simultaneously.With the optical radiation of 295nm is preferred, because it allows the tryptophan residue in the elicitor protein optionally, it can cause single or a limited number of disulfide linkage to destroy conversely.Be used for the spectrometer that the photoinduction disulfide linkage destroys, is suitable for including but not limited at certain proteic various light sources of wavelength region internal irradiation research grade, for example 75-W xenon arc lamp, deuterium lamp, the high voltage mercury lamp of RTC PTI spectrometer.Can obtain irradiation under the single wavelength by making light source and monochromator coupling.That single and source multiphoton excitation(MPE) comprises the high-peak power pulse or continuous wave (CW) laser apparatus.

Photoinduced disulfide linkage destroys the back and form new thiol group in albumen.Can be by based on 5,5 '-dithio is two-and the mensuration of (2-nitrobenzoic acid) [DTNB] measures its new life (denovo) outward appearance.With regard at,, detect a thiol group of the albumen formation of average each irradiation at 295nm place irradiation.In natural at, do not have free mercaptan, and the destruction that is adjacent to the disulphide of single tryptophan residue only produces a mercaptan accessible solvent, that can detect by this method.Will be at the photoinduced accessible thiol group that forms on the albumen in conjunction with the free sulphur alcohol radical on any mercaptan binding partner or the carrier.

This method that is coupled to carrier can be used for making up oligomer or the polymkeric substance that various types of disulphide connect.Can and comprise peptide or proteic carrier coupling with the photoinduced thiol group in given albumen or peptide or other biomolecules.As long as the concentration of albumen and carrier molecule is enough high in the linked reaction, crosslinked based on SS just will take place between adjacent molecule.When photoinduced albumen comprised the SS bridge, the aromatic moieties that has spatial neighbor was optional, because can be by adding aromatic moieties to linked reaction, maybe can simulate their compound and aromatics influence to this reaction is provided.

This photoinduced mercaptan and carrier link coupled method can be effectively applied to the carrier molecule of other type, the medicine on the pharmacopedics for example is so that promote it effectively to send.For example, make the water soluble molecules (including but not limited to peptide or albumen) that comprises disulfide linkage through photoinduction mercaptan and drug coupling can help water insoluble, be slightly soluble in the solvency action of water or hydrophobic drug and send.The molecule that is coupled to medicine in addition can be used to protect medicine away from its physiological environment, improves its body internal stability thus.This specific characteristic makes that the such labile drug of albumen is very attractive to this technology for for example sending.By implanting in the treatment site, the positioning delivery of the medicine of coupling molecule will reduce the general exposure of patient for medicine.Generally the prodrug that carrier is connected is defined as and comprises the prodrug that given active substance and Instantaneous Carrier group temporarily are connected, described carrier group can produce the physical chemistry or the pharmacokinetic properties of improvement, and can easily remove this carrier in vivo by the hydrolysis cutting usually.In the present invention, make the disulfide linkage of the light mediation of medicine link molecule destroy the controlled release that the molecule coupling form that can be used for from implant the patient realizes active medicine.This frequency that will make medicine be delivered to the patient minimizes, and the dispenser of light guide is provided.We also can make the delivery process optimization by only shining the body region that wherein must discharge medicine.These characteristics will be improved patient's conformability, especially for the medicine (for example defective of some albumen or metabolite) of chronic indication, the frequent injection of needs.Send with regard to medicine through skin, can be in the sphere of penetration of infrared light, induce the drug release of control by infrared light (by means of two-photon excitation), and bigger UV-light (or the infrared light that excites by means of three-photon) penetrates the promotion medicine is deeper discharged in the patient.Equally, as employed in PDT (photodynamic therapy), for example in the treatment of cancer/tumour patient, utilize optical fiber to make it possible in health, send light with the various degree of depth.Because the disulfide linkage that is exposed to solvent can destroy in the reductive environment, thus when the carrier with drug coupling enters the reducing environment in the tenuigenin gap of cell for example also releasable medicaments.

This photoinduction mercaptan link coupled method also can be used for proteopexy to upholder.The common type of the key that forms during being coupled to upholder is that disulfide linkage and sulphur-metallic bond (mainly are sulphur-Jin), wherein can form the layer of self-assembly.Because two types key all is stable, so washing completely can not replace this albumen after fixing.Can be by changing the intensity and the time length of protein concentration or ultraviolet light irradiation, and for example use L-the halfcystine, (reagent of 2-(2-pyridyl dithio) ethane amine (ethaneamine) hydrochloride (PDEA) or control proteic density (Hong Q. on the upholder subsequently with remaining activatory thiol group on mercaptan-lipid bilayer confining surface, Deng the people, 2001, Biochemical SocietyTransactions 29 (4): 587-582).Therefore sealing has the upholder of equally distributed ankyrin to prevent non-specific combination.According to the present invention, the fixed method does not comprise any chemical step, because the mercaptan activatory albumen spontaneously self-assembly on upholder that forms by ultraviolet radiation.The permutoid reaction of described mercaptan and disulphide is to make molecule be bonded to the method effectively and fast of upholder.Special benefits of the present invention be its avoided with albumen in the chemistry of free mercaptan produce relevant several shortcomings.As shown in Example 10, make some albumen, for example at inactivation by the reductive agent (DTT or beta-mercaptoethanol) that is used to produce free mercaptan.If use the reductive agent chemistry in albumen that comprises thiol group to produce free mercaptan, must before fixing, be removed so, during this step, can form disulfide linkage again.Alternative reductive agent for example lacks the 2-propyloic of thiol group) phosphine has the shortcoming with other radical reaction.Utilize reductive agent to destroy disulfide linkage in addition and also have the shortcoming that depends on pH about its chemical stability and reducing activity thereof.

But it is also proteic fixing on ground, the space control upholder.At present laser technology is allowed the focus with 1 micron or smaller szie.If will comprise special albumen or the target molecule and the upholder incubation that combines mercaptan of SS bridge, the formation of so photoinduced thiol group and coupling just can be limited to the focus of irradiation.Should control the viscosity of solution so that the diffusion process that may make the molecular dispersion of irradiation surpass spot size minimizes.This method will make discernible with different molecules to be deposited on the support surface very densely.Therefore method of the present invention can be used for loading microarray with molecule.

The present invention further aspect, can with homogeneous and repeatably mode control the direction of ankyrin.The selective excitation of the tryptophan residue that prolongs in albumen will only cause destroying the disulfide linkage that is transferred excitation energy.Thereby can form the position of free sulphur alcohol radical from the destructible disulfide linkage of proteic structure prediction light, wherein analyse the understanding protein structure from three-dimensional model, nucleus magnetic resonance (NMR) or X ray diffractive crystal credit.Only inducing by the tryptophan residue in the irradiation albumen in the situation of single thiol group, as situation at, then will be uniquely by means of this thiol group generation albumen fixing on upholder.Destroy with disulfide linkage and alternative approach that thiol group forms, for example utilize the method for reductive agent opposite, photoinduction method of the present invention causes the target destruction of disulfide linkage, thereby forms one or have only some can predict the accessible thiol group of its position exactly.Therefore fixed albumen will have single or very a limited number of direction subsequently.Because at has only the single thiol group away from avtive spot that is used for fixing, so by the fixing accessibility that can not limit substrate.The same with the situation of at and N,O-Diacetylmuramidase, by means of fixing proteic conformation of also unlikely change or structural performance away from the surperficial accessible thiol group in protein-active site.In other words, fixing means of the present invention can be used to be maintained fixed proteic native state.All functions/structure determination of carrying out on the albumen that is fixed with equidirectional according to the inventive method derives from generation the data of albumen homogeneous colony.The consistence of the proteic 26S Proteasome Structure and Function that is fixed and the maintenance of native state thereof have first importance for screening or measuring proteic catalysis, combination or any other biological characteristics, and one of many valuable advantages of the present invention are provided.The albumen that just has antimicrobial property, N,O-Diacetylmuramidase for example, can make described proteopexy (for example, food, skin, packing) to the surface is useful to prevent microorganism growth and to infect.

The present invention further aspect, can destroy making the key of proteopexy, thereby albumen is discharged in the solution to upholder.This is for disulfide linkage, and the mercaptan metallic bond all is possible.

Can use the identical mode of disulfide linkage on the albumen spatially contiguous, for example destroy disulfide linkage between albumen and the upholder with ultraviolet light irradiation with destroying aromatic amino acid wherein.Aromatic amino acid can be positioned at the albumen that is fixed originally on one's body, or with aromatic amino acid, for example the form of tryptophane solution provides, to be applied to support surface.Known light by about 254nm destroys disulphide bond (SS) itself.Alternatively, available (dithiothreitol (DTT)) DTT or well known to a person skilled in the art that other reductive agent destroys the disulfide linkage between albumen and the upholder.After retainingf key destroys, but the albumen that purifying discharges can be used it for further experiment in case of necessity.

The further aspect of the present invention is by using O ₂-plasma body (plasma) processing or Piranha remove through mercaptan Au key fixed albumen and make gold surface regeneration, remove comprising of gold surface of proteic top layer thus.

Embodiment

Embodiment 1: form the free thiol group after the UV-irradiation at

Utilize to separate from the at lipase/esterase characteristic of pea fusarium solanae and check after the UV-irradiation destruction of disulfide linkage in the albumen.

The stable state fluorescent emission intensity of at

Under following condition, make ultraviolet light irradiation that the at preparation is subjected to 295nm fluorescent emission intensity: in the big cuvette of quartz (1cm path length), in the cumulative period of the at mother liquor of 295nm Continuous irradiation 3ml 2 μ M (0 hour, 1 hour, 2 hours, 3 hours, 4 hours and 5 hours) with the stable state of following the trail of its Continuous irradiation.By providing the optical excitation of 295nm with the monochromator link coupled xenon arc lamp that provides by RTC 2000PTI spectrometer.Cuvette is installed in the constant-temperature sample pool, keeps 25 ℃ constant temperature.By making the at sample remain uniform solution with magnet 700rpm continuously stirring.To excite and launch slit (slit) and be set to 6nm.The at fluorescence intensity at monitoring continuously 350nm place from start to finish between 5 hours light period.Fig. 1 shown with after the 295nm rayed, in the fluorescent emission intensity of the time-dependent manner of the 2 μ M at solution at 350nm place.Very rapidly increasing in first 7200 seconds fluorescence intensities, is that fluorescence yield appears stable steady section then.

The detection of free mercaptan in the at after the UV-irradiation

The concentration of the free sulphur alcohol radical that forms in the at after following definite UV-irradiation: by based on as shown in Figure 2 thiol group and 5,5 '-dithio is two-reaction of (2-nitrobenzoic acid) [DTNB], or with reaction (Ellman GG, 1959 Arch.Biochem.Biophys.82:70-77 of EllmanShi reagent; Hu ML., 1994, Meth.Enzymology 233:380-385) spectrophotometry detects and the quantitative thiol group on the at.In the big cuvette of quartz (1cm path length), utilize the at solution different period of RTC 2000 PTI spectrometers as mentioned above with the 3ml 17.3 μ M of 295nm rayed in the TrisHCl that is present in 20mM pH8.5.Measure the fluorescent emission intensity of the time-dependent manner of 350nm place sample.All slits are set to the 2nm bandwidth.Before or after its 295nm irradiation, in the at solution of 900 μ l, add excessive DTNB (the 8.5mM DTNB mother liquor of 100 μ l in anhydrous methanol).DTNB mother liquor in the methyl alcohol is stablized maximum 2 weeks (Hu ML., 1994, see above) at 4 ℃.After mixing two kinds of compositions, use the Pharmacia spectrophotometer of UV-light/visible light immediately, measure NTB ion (nitro thiobenzoic acid salt ion, ε that the 412nm place discharges _412nm=13600M ^-1Cm ^-1) absorbancy, and after 20 and 24 fens clock times of 25 ℃ of reactions, measure once more.The absorbance at free concentrations of mercaptans and 412nm place is proportional.Reading is stable between 20 and 24 minutes.Each data point is the average of 3 observed values after 24 minutes.Control sample is included in the 8.5mMDTNB mother liquor of 100 μ l in the anhydrous methanol, its with 900 μ l not the at of irradiation (17.3 μ M at solution in 20mM TrisHCl pH8.5) mix.The concentration that improves the at sample of irradiation exceeds the limit of detection of DTNB method with the amount that guarantees the free sulphur alcohol radical that the irradiation back forms.

In non-irradiated at control sample, utilize DTNB to measure and do not detect the free thiol group.This is consistent with shortage free thiol group in natural at, because 4 all cysteine residues all participate in disulfide linkage in this enzyme.The amount of the thiol group that forms in the at sample between the light period is measured as the function that fluorescence intensity increases (F/Fo).Fig. 3 shows behind ultraviolet excitation in the increase of the time-dependent manner of the Trp of 295nm fluorescent emission intensity, and the positive correlation between the concentration that the fluorescent emission of different ratios increases (F/Fo) detected free sulphur alcohol radical increases.

These results support tryptophane (Trp) initial fluorescence intensity since near have complete disulfide linkage and the height quencher, and the increase of single endogenous Trp fluorescence intensity is because near the working model of the disulfide cleavage of at 295nm irradiation back in the at.

Embodiment 2: the destruction of radiation-induced disulfide linkage depends on the existence of the Trp residue of spatial neighbor in the natural at

In order to prove the effect of tryptophan residue in radiation-induced disulfide linkage destroys in the at, the Trp fluorescent emission intensity of native protein is all compared by the at that is reduced of chemical depletion with wherein all disulfide linkage.For the ease of reducing its all disulfide linkage, make the sex change of at protein part by heating.Use the Trp fluorescent emission intensity of the at of natural and sex change behind the irradiation to prove the spatially contiguous disulfide linkage of Trp residue, be used for excitation energy is transferred to from Trp the importance of disulfide linkage.

With the natural at of DTT reduction

In natural at, the disulfide linkage in folded protein is difficult near solvent, and it can not directly reduce by DTT.Yet, if the heating at is separated folding temperature to surpassing it in the damping fluid of pH8.5, separating folding process and will help DTT so subsequently near proteic disulfide linkage.Diluting soln at carries out the thermally denature step to avoid proteic precipitation.The damping fluid (20mM Tris-HCl 8.5) that selection is used for the at thermally denature shows that pH minimum with respect to temperature and volume change changes, and Tris-HCl has minimum ionizing event enthalpy in addition.From the OD 280nm of solution, utilize the optical extinction coefficient (13500M of at 280nm ^-1Cm ^-1) estimate the concentration of at solution.

The following disulfide linkage that carries out with DTT reduction at: the sample that 650 μ l is suspended in 1 μ M among the 20mMTrisHCl pH8.5 is heated to 70 ℃ from 25 ℃, adding excessive DTT (the 3.7M DTT in 20mM TrisHCl pH8.5 of 8.5 μ l) to it, is the DTT of 50mM to produce final concentration.

Trp fluorescent emission intensity natural or the reductive at is measured

Under following condition with RTC 2000 PTI spectrometer, measure after 295nm excites, the emmission spectrum of 1 μ M at sample, slit is set to the 2nm bandwidth: (A) when no DTT in 25 ℃ of following incubation at; (B) when no DTT in 70 ℃ of following incubation at; (C) be heated to 70 ℃ and be cooled to 25 ℃ after, when no DTT in 25 ℃ of following incubation at; (D) be heated to 70 ℃, after adding DTT and being cooled to 25 ℃, when DTT is arranged in 25 ℃ of following incubation at.Proofread and correct spectrum for the Raman base value.

4 kinds of emmission spectrum have been shown among Fig. 4.Emmission spectrum (A) in the at of 25 ℃ of incubations is identical with the at emmission spectrum (C) that is cooled to after 25 ℃ from 70 ℃, show the at of pH8.5 to add pyrolysis folding be the reversible process.This can compare with the emmission spectrum of the at of separating folded state (B).When having DTT, be heated to after 70 ℃ Trp emitting fluorescence intensity 25 ℃ at (D) greater than the natural at that does not have DTT in (A).As shown in embodiment 1, these spectrum are used for confirming the dependency between the reduction of the increase of Trp fluorescent emission intensity of at (exciting the back at 295nm) and its disulfide linkage.

Obtaining sample (A) and (C) in the process of the continuous wave spectrum after the continuous agitation of 295nm at Trp residue, observing fluorescent emission shown in embodiment 1 increases.Sample (A) and similar fluorescence (C) increase consistent with close proximity between observed Trp and the disulfide linkage in the three-dimensional structure after natural at folds again.After the continuous agitation of the Trp of 295nm at residue, for sample (B) or (D) do not observe this increase of fluorescent emission.The conclusion of these data supports is: the close proximity in the between Trp residue and the disulfide linkage is that photoinduction mechanism is necessary, and when at 70 ℃, when pH8.5 separates folded protein, has lost this degree of approach.This be likely at why at 70 ℃ Trp fluorescence quantum yield than in the high reason of 25 ℃ quantum yield, although collisional quenching produces lower quantum yield between Trp and solvent molecule owing to higher temperature causes in expection.

Photoinduced disulfide linkage destructive specificity in embodiment 3. albumen

Made the at transgenation of pea fusarium solanae, with the at polypeptide of encoding mutant, wherein the amino acid alanine by non-fluorescence substitutes one tryptophan residue (W69A).Below the proof mutant is compared with natural at and is lacked photoinduced disulfide linkage destruction, further need to confirm the aromatic amino acid (for example trp) of closely adjacent disulfide linkage on the space.The at of recombinant expressed sudden change (W69A).The mutant or the native protein solution that will be present in the 3ml2 μ M among the 50mM Tris-HCl pH7.0 change cuvette over to, at 25 ℃ of constant temperature, carry out magnetic agitation with 700rpm, be used to shine at 296nm place, use 10 and the exciting and launch slit of 2nm respectively from the spectrophotometric 75W xenon lamp of RTC 2000 PTI that is coupled to monochromator.As shown in Figure 5, can find out from the absorption (A) and the emmission spectrum (F) of aromatic amino acid the aqueous solution of pH7, tryptophane be unique be the light activated aromatic moieties of 296nm by wavelength.Before or after 296nm irradiation, be that the intramolecularly quenching probe BODIPY FL L-Gelucystine (Fig. 6) of 20 μ M is in the dark 25 ℃ of incubations 20 minutes with mutant or natural at solution (1ml) and final concentration.(excitation wavelength of BODIPY FLL-Gelucystine) excites every kind of at sample that comprises BODIPY FL L-Gelucystine at the 480nm place then, and write down 500 and 620nm between emmission spectrum, wherein utilize respectively to be set to 4 and the exciting and launch slit of 2nm.Any thiol group that between the light period, produces in the at sample will with the SS radical reaction of probe Bodipy FL L-Gelucystine (comprising 2 bodipy groups), cause the destruction of this molecule and increase by 2 distances between the bodipy group subsequently.This causes the increase of each bodipy group fluorescent emission intensity conversely.As shown in Figure 7, opposite with natural at (B), the irradiation of sudden change at (A) does not produce the Bodipy fluorescence of increase, and showing does not have thiol group to form in mutant.This shows that after the 296nm irradiation disulfide linkage that is adjacent to the W69A sudden change in the sudden change at is kept perfectly owing to lack the Trp residue in the tight quarters degree of approach.

By further studies confirm that the characteristic of destructive disulfide linkage in the at natural after the UV-irradiation.Known natural at has 2 disulfide linkage, and one of them approaches avtive spot, and its integrity is absolutely necessary to catalytic activity, and second away from avtive spot, and approaches the Trp residue.Because find that natural at keeps its enzymic activity after UV-irradiation, so clearly have only the disulfide linkage of surface localization to destroy.People such as Prompers, 1999, the NMR of FEBS Lett.456:409-16 and mass-spectrometric data show that also UV-irradiation destructive SS bridge is a bridge that approaches the Trp residue.Behind the ultraviolet light irradiation in the natural at destruction that forms quantitative disulfide linkage of the detectable thiol group of DTNB other instrument is provided.Each at molecule only detects a thiol group after the UV-irradiation.This destruction with the disulfide linkage of surface localization is consistent, wherein second not accessible solvent of thiol group.The accessible solvent of at least one cystine residue of second disulfide linkage, but do not detect destruction.

Embodiment 4. predictions have the albumen of the derivable Trp-SS triplet of light

In the albumen database (Protein Data Bank) of Research Collaboration for Structural Bioinformatics (RCSB), identified the albumen that comprises disulfide linkage and aromatic moieties; wherein selected (trp) residue indoles nuclear carbon or nitrogen-atoms are positioned at 5 of disulfide linkage S atom or less than 5 (people 2000 such as Bergman; Nucleic AcidsResearch, 28:235-242).Only consider the albumen of those known NMR or x-ray crystal structure.Utilize molecule video picture program: the distance (Guex, N and Peitsch, M.C., 1997, Electrphoresis 18:2714-2723) between the DeepView/Swiss-Pdb of www.expasy.org.spdbv Viewer measurement triplet atom.Mate selected albumen at having the albumen sorted lists that is less than 90% sequence identity and belongs to each different enzyme class [EC numbering and classification].For the purpose of analyzing, do not consider that trp-SS triplet wherein is arranged in the albumen (lytic enzyme) of part.In addition, because molecule additional or repeat in the molecule dimer, tripolymer, unit cell, so once triplet in the PDB clauses and subclauses, occurs surpassing only as once considering.

Discovery Trp-SS triplet in the middle of lytic enzyme is general, and at is the member of described lytic enzyme.According to its characteristic to 8 , 12 that are located at the Trp-SS triplet of identifying in every kind of these albumen with the amino-acid residue in the 15 radiuses calculates and classify (wherein the spherical regional center is to approach the indole ring of the tryptophane of disulfide linkage most): Gly, Ala, Val (short aliphatic series); Leu, Ile (long aliphatic series); Pro; Ser, Thr (comprising hydroxyl); Cys, Met (sulfur-bearing); Asn, Gln (acid amides); Asp, Glu (tart); His, Lys, Arg (alkalescence); Phe, Tyr, Trp (aromatics).Generally speaking by Mathews, Christopher K. and van Holde, K.E., in " Biochemistry " second edition, The Benjamin/Cummings PublishingCompany, Inc., 1996, ISBN:0-8053-3931-0 has determined every group of amino acid whose average frequency of occurrences in the albumen, and it is 23.4% (Gly, Ala, Val); 12.1% (Leu, Ile); 4.6% (Pro); (13.1 Ser, Thr); (4.5 Cys, Met); 8.3% (Asn, Gln); 11.7% (Asp, Glu); 13.8% (His, Lys, Arg) and 8.1% (Phe, Tyr, Trp).

Count the occurrence rate and the classification of every kind of residue according to the characteristic of residue.Deduct 1 tryptophane by number, and remove influence from the triplet residue from deduct 2 halfcystines from the occurrence rate number the residue that comprises sulphur of every kind of spherical shape residue tabulation from aromatic moieties.Following then calculating is for the frequency of occurrences of each group in subgroup, f (subgroup):

Total residue number in the number/subgroup of residue in formula 2.1 f (subgroup)=this group.

Use f (subgroup) to calculate the mark score value, its be the frequency of occurrences in the subgroup divided by the frequency of occurrences total in the albumen (referring to table 2.1):

Formula 2.2 score values=f (subgroup)/f (albumen)

Therefore: " score value "＞1 is meant that residue/group was performance

" score value "＜1 is meant that residue/group owes to show

For every kind of triplet, behind the score value by every kind of residue of row arrangement in spreadsheet, utilize the relevancy tool calculating triplet of data analysis tool bag and the similarity of a kind of at (W69, S31, S109).This operation produces relation conefficient, and it is the measuring of similarity between the triplet of at and the triplet (n).

Following calculating relation conefficient:

Formula 2.3

T[cut wherein] and t[n] represent the triplet and the triplet n of at respectively, and be the standard deviation of the triplet considered.

Use relation conefficient triplet to be classified with regard to its similarity with respect at.Contour plot with score value carries out graph-based to the result, wherein lack the residue group that occurs less than 1 branch value representation than expection, is equal to or higher than that 1 branch value representation occurs with expected frequence and with the residue group of the frequency appearance that is higher than expection.

Especially in 8 radiuses, identify the special subgroup of amino-acid residue, it obviously is different from the amino acid whose average frequency of occurrences in the common albumen, particularly spends about half of following residue: (Phe, Tyr, Trp) (Asp, Glu) (Asn, Gln) and Pro (Fig. 9).Therefore, amide residues (Asn, Gln) was performance, and the occurrence rate of in most of the cases short aliphatic residue (Gly, Ala, Val) and/or long aliphatic residue (Leu, Ile) also was performance.In each triplet almost, there are the charged amino acid (His, Lys, Arg) (Asp, Glu) owing to show and the group of Pro residue.

The special subgroup of seeing in 8 radiuses of triplet of conserved amino acid is not as obvious in 12 and 15 .Amino-acid residue analysis in the immunoglobulin (Ig) in the trp-SS triplet 8 radius degrees of approach has shown that comprising the amino acid whose of hydroxyl crosses performance, and aromatic moieties also was performance in many IgG TrpS-S triplets.In the amidoamino acid in many immunoglobulin (Ig)s (Asn, Gln) one or both and aliphatic residue occurrence rate also all were performances, and wherein the amino acid around the triplet of TrpSS space is formed the composition (immunoglobulin part of IGG1 triplet A group) that is very similar to around the at triplet.We also observe the charged amino acid (His, Lys, Arg) (Asp, Glu) owing to show and the group (Figure 11 and 12) of Pro residue in these immunoglobulin (Ig)s.Wherein the emission dipole plane of aromatics Trp side chain unlikely exchanges excitation energy perpendicular to those Trp-SS triplets of disulfide linkage absorption dipole planar between excited state Trp and disulfide linkage.

But check some members' of this histone light inducing properties, described albumen comprises and similar Trp-SS triplet and the amino-acid residue subgroup found at Trp-SS triplet 8 radiuses.Utilize intramolecularly quenching probe BODIPY FL L-Gelucystine (Fig. 6), described as embodiment 3 for the mutant at, measure every kind of derivable disulfide linkage of proteic light and destroy.Shown in Fig. 8 A-F, following albumen: hen egg-white lysozyme, snow-white head mold TGL, human plasminogen, placental alkaline phosphatase, rennin B and human normal immunoglobulin all demonstration comprise light-derivable Trp-SS triplet.

The albumen alpha-lactalbumin comprises 4 disulfide linkage, wherein has only 2 to destroy when UV-irradiation, i.e. Cys6-Cys120 and Cys73-Cys91 (people (2002) Biochemistry such as Vanhooren, 41 (36): 11035-11043).Although Cys73-Cys91 and Cys28-Cys11 all have the sulphur atom that at least one is positioned at nearest atom 4 of tryptophane indole ring in the alpha-lactalbumin, have only the former responsive to photoinduced destruction.Trp118-Cys28-Cys111 is opposite with triplet, triplet Trp60-Cys73-Cys9 is positioned at spheroidal zone 8 radiuses, its amino acid is formed similar to the at triplet, particularly about the abundance of following residue: (Phe, Tyr, Trp), (Asp, Glu), (Asn, Gln) and Pro residue (Figure 10).

Embodiment 5. photoinduced at couplings.

In embodiment 1,2 and 3, shown from replying irradiation in the pattern albumen at of pea fusarium solanae and other the several albumen and formed free SH group.Then the reactive thiol group of at can with the carrier molecule coupling in the solution, described carrier molecule has the thiol group of free and accessible solvent or the disulfide linkage of accessible solvent.As shown in Fig. 2 and 6, be coupled to the method for carrier molecule in the solution by induce Mingguang City's inductive at the disulfide group coupling that makes free thiol group and DTNB and BODIPY FL L-Gelucystine in the at the photoirradiation of 295nm.In embodiment 3 (Fig. 8), also proved the photoinduction coupling between each in BODIPY FLL-Gelucystine and albumen lysozyme, lipase, Profibrinolysin, alkaline phosphatase, rennin, the immunoglobulin (Ig).

The following linked reaction of carrying out.Described as embodiment 1 and 2, utilize RTC 2000 PTI spectrometers, in the big cuvette of quartz (1cm path length), use the 295nm 3ml 17.3 μ M at solution different periods of rayed in 20mMTrisHCl pH8.5.Before or after the 295nm irradiation, excessive DTNB (being dissolved in 100 μ l 8.5mMDTNB mother liquors in the anhydrous methanol) is added in the 900 μ l at solution.After mixing two kinds of components, use NTB ionic absorbancy (nitro thiobenzoic acid salt ion, the ε of UV-light/visible light Pharmacia spectrophotometer measurement 412nm place release immediately _412nm=13600M ^-1Cm ^-1), and after 25 ℃ 20 and 24 minute reaction times, measure once more.Cause forming the NTB ion of stoichiometric quantity with the linked reaction of DTNB.Therefore, the absorbance at the concentration of link coupled at and 412nm place is proportional.Control sample comprises the 100 μ l 8.5mMDTNB mother liquors that are dissolved in the anhydrous methanol, its with 900 μ l not the at of irradiation (being dissolved in 17.3 μ M at solution among the 20mM Tris-HCl pH8.5) mix.

Embodiment 6. photoinduced proteopexies.

The reactive thiol group (as shown in embodiment 1,2 and 3) of free of in albumen for example, replying irradiation and forming from the at of pea fusarium solanae, can be used for albumen is connected with the upholder or the carrier molecule of thiol-reactive, no matter it is a gold or with the gold of thiol group derivatize, or with the quartz surfaces of SH group derivatize, or with the polymkeric substance upholder or the carrier molecule of SH group derivatize.Fixed albumen shows maintenance its functional performance (for example enzymic activity).

At first use the SH group with polymkeric substance upholder derivatize by using the NHS/EDC that carboxymethyl is modified to the inferior fat ester of N-hydroxyl succinyl-to activate.Then by with the borate buffer solution that is dissolved in 0.1M pH8.0 in the cysteamine incubation, and subsequently with the borate buffer solution that is dissolved in 0.1M pH8.0 in DTT or DTE (dithioerythritol) incubation and on upholder, introduce thiol group.Borate buffer solution with 0.1M pH8.0 before fixing washes upholder.

Albumen is connected with quartzy upholder with SH group derivatize, and as being the example explanation with many albumen, it is fixing to utilize follow procedure to pass through the monitoring of total internal reflection fluorescent (TIRF) spectroscopy:

Step 1: the surface preparation that is used for the quartzy slide glass of TIRF spectroscopy

Cleaned quartzy slide glass (12cm by immersing chromic acid sulfuric acid (chromosulfuric acid) in 1 hour in (Merck1.02499/Z624399) at 70-75 ℃ ²), then in room temperature rinsing in water.Then by be dissolved in 5w/v% Potassium Persulphate (99% K in the deionized water in 99-100 ℃ of immersion ₂S ₂O ₈, Acros Organics 20201-000) in 1 hour, make the slide glass hydroxylation with the number that increases the OH group.With the hydroxylated slide glass of rinsed with deionized water and dry rapidly.

Then by being dissolved in m-xylene (99+%, Acros Organics 1808600100) 150 μ l 0.03%v/v 3-mercapto alcohol radical propyl group-Trimethoxy silanes (Merck 63800) in are applied to hydroxylated slide glass, prepare SH-activatory slide glass.Xylene solvent is evaporated fully from slide glass, use pure dimethylbenzene then; Ethanol and deionized water wash the surface in succession, and final drying produces the silane coating of uniform optics coideal.

Step 2: by TIRF spectroscopy with proteopexy on quartzy slide glass

With the SH-activatory or contrast hydroxylated slide glass and be installed on the quartz prism, be attached to flow chamber with the urethane packing ring of 10 μ m, and be assemblied in and be coupled to spectrophotofluorometer, provide in the TIRF instrument of 75W xenon arc light source with glycerine.By with 0.25ml ^-1Flow velocity flushing flow chamber or carry out proteic fixing with following solution incubation:

A) 25mM Tris-HCL, pH8.5 (buffer A) handled 5-10 minute, made the slide glass hydrated.

B) 25 ℃, be dissolved in the 0.5-2.0 μ M protein solution in the buffer A, handled 10-20 minute.

C) buffer A was handled 10 minutes.

D) nonionic detergent, for example 2v/v% Helmanex II (Hellma GmbH ﹠amp; CoKH, Germany), handled 5 minutes.

E) buffer A

The successive UV-irradiation, 280 or the condition of the UV-irradiation that limits of 296nm place (10 minutes light, or 1 second light/minute) or dark (contrast) under carry out proteopexy, and monitor by the fluorescent emission at 330nm place.Before measuring the fixed protein-active, use ethanol and H ₂O rinsing slide glass.

In the improvement of aforesaid method, the ultraviolet light irradiation protein sample, 2 μ M at solution for example are subsequently by with the sample incubation and/or be fixed on sample wash on the quartzy slide glass of SH derivatize, clean with buffer A again, and randomly remove unconjugated albumen with stain remover.

Step 3: fixing active enzymic activity

After the fixing step, the luciferase substrate is applied to quartzy slide glass, and by fluorescence spectroscopy detection reaction product.

A. UV light-induced at is fixed

As shown in figure 13, by monitoring at is being fixed under the irradiation of successive or qualification on SH activation and the hydroxylated quartzy slide glass at 330nm detection fluorescence.Flushing flow chamber (A-B) is then with 2.0 μ M at solution incubation (B-C); With buffer A rinsing (C-D), and with stain remover and buffer A rinsing (E).Between with damping fluid and stain remover flush period, remove non-combining closely to the at of quartzy slide glass.The at fixed relative efficiency of inferring from fluorescent emission intensity is big than under the irradiation that is limiting under the Continuous irradiation, and this depends on SH-activatory quartz surfaces (Figure 14 A) and shines (Figure 14 B).Limiting the risk that fixedly can be used under the optical condition reduces the opalescence bleaching.Do not show that the derivable disulfide linkage destructive sudden change at of bright dipping (W69A) can not be fixed on the quartzy slide glass of SH-activatory (Figure 14 B) under the same conditions.

According to the following step, by measuring the ester bond cutting of non-fluorogenic substrate butyric acid 4-methyl umbrella shape ester, the release that is accompanied by the fluorescence 4-methyl umbelliferone detects the enzymic activity that is fixed on the on the quartzy slide glass:

A) deposition is dissolved in 25mM Tris-HCl, the 10 μ M butyric acid 4-methyl umbrella shape ester substrates of the 25-100 μ l among the pH8.0 on quartzy slide surface.

B) this slide glass of incubation 30 minutes at room temperature.

C) by behind the digital photographing machine monitoring ultraviolet excitation (365nm), the fluorescent emission intensity of slide glass (450nm).(alternatively reaction mixture can be changed over to micro titer plate well and monitor fluorescent emission by the UV/VIS spectrophotometer.)

With substrate, butyric acid 4-methyl umbrella shape ester is dissolved in DMSO, produces the mother liquor of 25.2mM, it is added measure damping fluid.

Detect activatory at (Figure 15) on quartzy slide glass of SH-activatory rather than hydroxylated quartzy slide glass, this confirms that further the photoinduction coupling of at and the quartzy upholder of SH-activatory is the observation by means of thiol group.

B. fixing of UV light-induced N,O-Diacetylmuramidase and rennin

As above described at, 2 μ M N,O-Diacetylmuramidases or rennin solns be fixed on the quartzy slide glass and by TIRF spectroscopy monitor.What show N,O-Diacetylmuramidase fixedly is SH-activation photoactivation and that depend on quartzy slide glass (Figure 16 A is to B).If find that Continuous irradiation is defined in 10 minutes just can strengthen N,O-Diacetylmuramidase fixed coupling efficiency.Find similarly rennin fixedly be photoactivation and depend on coupling (Figure 16 C) with the SH-quartz surfaces.

The detection of lysozyme activity: detect the lysozyme activity that is fixed on the quartzy slide glass according to being used for detecting the active step of at, just except using the specific substrate of N,O-Diacetylmuramidase (EnzCheck Lysozyme, 22013, Molecular probes Inc), and with spectroscopy write down fluorescent signal.The 50 μ g/ml substrates (being dissolved among 0.1M phosphoric acid/0.1M NaCl pH7.5) of one 125 μ L are applied on the quartzy slide glass., 100 μ L solution are changed in the hole in black porous plate (96 holes, Nunc 267742) after 30 minutes at room temperature (20-25 ℃) incubation.In fluorescence porous plate reader (SpectraMax Gemini XS), in the 495nm excited sample, and the fluorescent emission intensity (FEI) at record 525nm place.Concentration and flow process all are based on from the scheme of the EnzCheck Lysozyme determination of activity test kit 22013 of Molecular probes Inc.

The lysozyme activity analysis shows also that compared with the control bigger with the activity on the SH-activation slide glass of UV-light-irradiation, described contrast comprises fixing/absorbing on the SH-activation slide glass or on the hydroxylation slide glass of UV-irradiation in the dark.

With EnzCheck Protease test kit E6639 (Molecular Probes Inc.), utilize the 10 μ g/mL concentration of substrate that are dissolved among the 10mM Tris pH7.8 (damping fluid that comprises in the test kit) to carry out the detection of milk-curdling activity.The substrate of one of 125 μ L is applied on the quartzy slide glass.After 30 fens clock times of room temperature (20-25 ℃) incubation, 100 μ L solution are changed in the hole in black porous plate (96 holes, Nunc 267742).In fluorescence porous plate reader (SpectraMax Gemini XS), in the 589nm excited sample, and the fluorescent emission intensity (FEI) at record 625nm place.Concentration and flow process all are based on from the scheme of the EnzCheck Protease determination of activity test kit E6639 of Molecularprobes Inc.

C. fixing of UV light-induced immunoglobulin (Ig)

Produce 2 F (ab) fragment with papoid proteolytic digestion immunoglobulin IgG mixture, every kind comprises 2 polypeptide.The three-dimensional structure of the existing segmental structural domain of IgG IgF ab shows that they all are rich in the Trp/Cys-Cys triplet.The existing segmental three-dimensional structure of Fab shows and is present in C _H, V _H, C _LAnd V _LThe not accessible solvent of disulfide linkage in the structural domain in the structural domain.Disulfide linkage is present in the Fab fragment base portion that is positioned at away from antigen-binding site between other structural domain, connects conserved regions.Near the accessible solvent of this disulfide linkage and being positioned at the Trp residue.

Can pass through the destruction of UV-light-inductive F (ab) fragment disulfide linkage, the destruction that for example is positioned at the disulfide linkage (Cys136-Cys214) of Trp 196 10 -11 between the C-end structure territory of 1CBV mouse IgG1 F (ab) realizes that the orientation of F (ab) fragment on the upholder of SH-group derivatize fix.Be positioned at another terminal antigen-binding site of Fab fragment away from fixing site, therefore keep its validity for antigen recognition.

The segmental purifying of the following step explanation F (ab), its UV light-induced fixing and be coupled to upholder the segmental detection of the active F of function (ab) arranged:

A) according to people such as Aybay 2003, the method of Immunology Letters 85:231-235 digests mouse IgG1 (Sigma M7894) with papoid (Sigma P4762), and can utilize Biotechnology by Pierce, ImmunoPure F (ab) test kit purifying F (ab) fragment that Illinois, the U.S. provide.

B) will be dissolved in 25mM Tris-HCl by means of the UV-irradiation of 280nm to 296nm wavelength as mentioned above, 1-10 μ M F (ab) the fragment solution among the pH8.0 is fixed on the quartzy slide glass of SH-activatory in the TIRF instrument flow pond.Aromatic amino acid can be included in the fixing damping fluid so that photoinduction is kept off between the protein structure domain of endogenous aromatic amino acid or the destruction of disulfide linkage in the structural domain, also it can be used for fixing effectively IgF (ab) fragment.By in the dark and on hydroxylated upholder, contrast the fixing link coupled specificity of testing.The fluorescent emission at the 330nm place that excites at the 296nm place provides being fixed on the measurement of the F (ab) on the slide glass.

C) by will in Tris-HCl pH8.0, dilute 100 * anti--specific fluorescein isothiocyanate conjugate of mouse IgGF (ab) (Sigma F2653) import flow cell, to determine the segmental antigen-binding activity of fixed F (ab) in step (b).After 25 ℃ of incubation 10-30 minutes, by removing unconjugated conjugate, then in the fluorescent emission intensity that is bonded to the conjugate of quartzy slide glass in the 525nm detection that excites at 495nm place with buffer A flushing flow cell.

D. UV-light-inductive notatin is fixing

Use the β-D-glucose in the notatin quantitative liquid (for example human blood).Notatin comprises the trp-SS triplet (Cys164-Cys206) from Trp133 7-9 of being positioned at of accessible solvent, and the photoinduced destruction of this disulfide linkage produces the thiol group that is adapted for coupling to carrier.Because the position of disulfide linkage is away from avtive spot, so its destruction and the mercaptan that produces subsequently are coupled to the catalytic activity that upholder can not slacken notatin.Description at is fixed on 2 μ M notatin solution on the quartzy slide glass as mentioned, and monitors by TIRF spectroscopy.

The proteopexy of embodiment 7. spatial control

The at molecule is fixed on the quartzy slide glass of SH-activatory, with its space be limited in 1-2 μ m ²The zone in.With a droplet be dissolved in 5 μ M at solution among the 25mM Tris-HCl pH8.5 in room temperature deposition on the quartzy slide glass of SH-activatory.With the laser radiation droplet of 296nm, the tip of laser probe is immersed in the droplet and is positioned at the quartzy surface 1-2mm of activation thus.The at of utilizing embodiment 6A to provide is measured, and detects fixed at activity in the zone that the space limits on slide surface.

Embodiment 8. uses the reversible proteopexy of UV-light

UV-light is used to discharge by the photoinduced albumen that is incorporated into the thiol-reactive surface with disulfide linkage, for example at of fixing.When tryptophan residue and disulfide linkage existed in solution, the irradiation tryptophane destroyed disulfide linkage by the mechanism of photon induced.Discharge the fixed at according to the following step by ultraviolet light irradiation:

1. the slide glass that will be fixed with at is placed in the TIRF equipment and the protein concentration by adsorbing on the fluorescent emission monitoring slide glass at 350nm place.

2. with the 20mM Tris-HCl pH8.5 damping fluid flushing slide glass of the compound that comprises 20 μ M tryptophanes or aromatic amino acid residue or simulation aromatic moieties of continuous flow.

3. use 295nm photoirradiation slide glass 5 hours.

4. with 20mM Tris-HCl pH8.5 buffer solution for cleaning slide glass.

5. measure the fluorescence of slide glass behind the albumen wash-out continuously at the 350nm place.

Embodiment 9. uses the reversible fixing of reductive agent

By the chemical reduction of disulfide linkage, discharge through photoinduced fixing by the at of disulfide-bonded to the thiol-reactive surface.After this, in not containing the 20mM Tris-HCl pH8.5 damping fluid of reductive agent, dialyse albumen to recover its natural structure.Discharge the fixed at according to the following step by reductive agent:

1. the slide glass that will be fixed with at is placed in the TIRF equipment and the protein concentration by adsorbing on the fluorescence monitoring slide glass at 350nm place.

2. the damping fluid (the 20mM Tris-HClpH8.5 that contains 50mM DTT) with continuous flow cleaned slide glass 30 minutes, 1 hour or maximum 5 hours.

3. with 20mM Tris-HCl pH8.5 buffer solution for cleaning slide glass.

4. measure the fluorescence of slide glass behind the albumen wash-out continuously at the 350nm place.

Embodiment 10: photoirradiation or reductive agent are to the influence of protein function and stability

A. photoirradiation is to the influence of cutin enzymatic activity

Be placed in dark or in the light of normal (synthetical) laboratory the about 3 hours at least at most protein samples of irradiation compare, show at the protein sample (at) of 296nm irradiation and reduce active (Figure 17 A).

Irradiation at (1 μ M is dissolved among the 25mM Tris pH8.5) in PTI spectrofluorometer (excite slit to be set to 6nm, and the emission slit being set to 1/2nm).The 3ml sample is placed quartz cuvette, during 296nm place (in the record emission of 330nm place) Continuous irradiation, stir (500rpm, 7 * 2mm magnetic rod) constantly.Between the whole light period, collect 5 μ L samples and be used for instant activity measurement (in 5-10 minute).Control sample is placed on the testing table under the source of artificial light (neon lamp), and with the sample that places dark as a reference.

Measure the activity (in duplicate) of all 3 samples.For the purpose that contrasts, also analyze the pure substrate in damping fluid.

100 μ l substrate solutions (10 μ M butyric acid 4-methyl umbrella shape esters, Fluka 19362, are dissolved among the 25mM Tris pH8.0) are sucked in 8 holes of black porous plate (96 holes, Nunc 267742).In fluorescence porous plate reader (SpectraMax Gemini XS), in 365nm place excited sample, and during initial 8-10 minute every the fluorescent emission intensity (FI) of 1 minute record 450nm place product.(during initial 10 minutes, fluorescent emission intensity linearly increases r with the reaction times as the measuring of protein-active to use moment of fluorescence to increase (the Δ FI of per minute) ²＞0.98).The activity of report be in the activity of sample under the UV-irradiation or the artificial light control sample activity divided by the ratio between the activity that is placed in the reference sample in the dark.Measure active down at 25 ℃+/-0.1 ℃.DTT is to the influence of cutin enzymic activity

B. reductive agent is to the influence of cutin enzymatic activity

With the 30 μ M at period different with 5.1mM DTT incubation together.Showed among Figure 17 B that at is to the active experimental result of the time-dependent manner of tributyrin when having DTT.As seen at keeps this specific activity above 26 hours when not having DTT.When having DTT, with the ratio of 30 μ M to 5.1mM DTT, the at specific activity is reduced to 41% of initial activity at incubation during 45 minutes.Behind the incubation 4 hours, specific activity is 21% of an initial activity.

Claims (29)

1. one kind makes albumen or peptide and the carrier link coupled method that contains disulfide linkage, comprise the following steps,

A) irradiation albumen or peptide with the destruction by disulfide linkage in albumen or peptide, produce thiol group and

B) with the albumen of irradiation or peptide with carrier incubation that can the combined sulfur alcohol radical, obtain coupling thus, or

A) with albumen or peptide with carrier incubation that can the combined sulfur alcohol radical and

B) irradiation albumen or peptide when having described carrier produce thiol group with the destruction by disulfide linkage in albumen or peptide, obtain coupling thus.

2. according to the process of claim 1 wherein that described irradiation steps comprises the light of the wavelength that excites one or more aromatic amino acids.

3. according to the method for claim 2, wherein said aromatic amino acid comprises tryptophane, tyrosine and phenylalanine.

4. according to the method for claim 2 or 3, wherein said irradiation comprises that wavelength is the light of about 295nm, 275nm or 254nm.

5. according to the method for claim 3, wherein said aromatic amino acid is a tryptophane.

6. according to any one method in the claim 2,3 or 5, its medium wavelength is about 295nm.

7. according to any one method in the claim 1 to 6, wherein described albumen of irradiation or peptide when having the free aromatic amino acid.

8. according to any one method in the claim 1 to 7, wherein said carrier comprises peptide, albumen or biomolecules.

9. according to any one method in the claim 1 to 7, wherein said carrier is a upholder.

10. method according to Claim 8, wherein said coupling is to be fixed on the described upholder.

11. according to the method for claim 10, the wherein said spatial control that fixedly is subjected to.

12. according to the method for claim 10, wherein said upholder comprises gold.

13. according to the method for claim 10, wherein said upholder be can the combined sulfur alcohol radical the derivatize upholder.

14. according to the method for claim 10, wherein said upholder comprises thiol group or disulfide linkage.

15. according to the method for claim 14, wherein upholder comprises transcribed spacer.

16. comprise carrier by any one one or more albumen of method link coupled or peptide in the claim 1 to 15.

17. according to the carrier of claim 16, wherein said carrier is a upholder.

18. according to the carrier of claim 17, wherein said upholder is selected from electronic chip, slide glass, wafer, resin, hole, pipe, microarray and film.

19. carrier according to claim 18, wherein said upholder comprises the material that is selected from topaz, polystyrene, polyethylene, polyester, polyetherimide, polypropylene, polycarbonate, polysulfones, polymethylmethacrylate, poly(vinylidene fluoride), siloxanes, diamond, quartz and silica, silicon, metal, nylon, soluble cotton, agarose, Mierocrystalline cellulose and pottery.

20. according to any one carrier in the claim 16 to 19, wherein one or more albumen or peptide are selected from enzyme, transcription factor, protein structure domain, conjugated protein, antigen and immunoglobulin (Ig).

21. according to the carrier of claim 20, wherein said immunoglobulin (Ig) is F (ab) fragment.

22. according to the carrier of claim 20, wherein said enzyme is selected from, rennin, notatin, lipase, N,O-Diacetylmuramidase, alkaline phosphatase and Profibrinolysin.

23. according to the carrier of claim 16, wherein said carrier comprises the medicine on the pharmacopedics.

24. be used for the purposes of biological function reaction according to the carrier of any one in the claim 16 to 22.

25. according to the purposes of the carrier of claim 24, wherein said biological function reaction is selected from biosensor, chromatography, immunodetection, enzymatic determination, Nucleotide are in conjunction with detection, protein-protein interaction, protein modified, carrier target and targeting proteins.

26. be used to produce the purposes of biosensor or albumen/peptide microarray according to the method for any one in the claim 1 to 15.

27. be used to diagnose or be used for the purposes of biosensor test kit according to the carrier of any one in the claim 16 to 23.

28. prediction can destructive contains the method for the albumen or the peptide of disulfide linkage by irradiation, comprises step:

A) identify and select to contain the albumen of disulfide linkage or peptide and

B) identify and be chosen in selected albumen or peptide in (a), it further comprises the aromatic amino acid residue in 10 of described disulfide linkage, and

C) identify and be chosen in selected albumen or peptide in (b), the side chain dipole plane of wherein said aromatic amino acid and described disulfide linkage plane are not vertical.

29., further comprise step according to the method for claim 28:

Identify and be chosen in (b) or (c) in selected albumen or peptide, the amino-acid residue that wherein is positioned at described aromatic amino acid residue indole ring 8 radiuses is crossed the amidoamino acid residue (Asn, Gln) of at least 1 times of performance, and owe to show at least 1 times charged amino acid (His, Lys, Arg) (Asp, Glu) and proline residue and short aliphatic amino acid residue (Gly, Ala, Va1)) and/or long aliphatic amino acid residue (Leu, Ile).

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