CN103630518B - A kind of new method of the fluorescence probe detection hydrogen sulfide synthase activity of use hydrogen sulfide and its application - Google Patents
A kind of new method of the fluorescence probe detection hydrogen sulfide synthase activity of use hydrogen sulfide and its application Download PDFInfo
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Abstract
The invention belongs to technical field of biological, the fluorescence probe for being related to a kind of use hydrogen sulfide detects the new method of hydrogen sulfide synthase activity, and provides the application approach of this method.Include CBS, CSE and CL activity suitable for detection hydrogen sulfide synzyme, for tissue sample, first it is cut into small pieces and grinds again, for cell, histiocyte lysate is cracked, and pyrolysis product carries out protein quantification, such as schemes, reaction system (histocyte pyrolysis product, L cysteines, 5 phosphopyridoxal pyridoxal phosphates) is added in the reaction tank of reaction bulb outer ring, the absorption system (H of the adjuvant Cetrimide containing reaction2S fluorescence probes working solution) it is added in the absorption cell of inner ring, reaction system produces H2S gas molecules are combined with the fluorescence probe of absorption system, available for fluoremetry and fluorescence imaging, quantitatively detect H2S contents, more intuitively reflect H2The activity of S synzyme.The present invention can be research H2Signal path and H that S-phase is closed2Meanings and diagnosis of the S during some diseases provide preferable research method.
Description
First, technical field
The invention belongs to technical field of biological, and in particular to a kind of fluorescence probe detection hydrogen sulfide of use hydrogen sulfide is closed
New method and its application into enzymatic activity.This fluorescence probe detection enzymatic activity method can be important for some in research histocyte
Mechanism during zymetology, illustrates some important signal paths in biosystem, be some major diseases early diagnosis and
Prevention provides good supplementary means.
Two background technologies
For a long time, hydrogen sulfide (hydrogen sulfide, H2S) it is considered as a kind of poisonous gas, however, with
To H2Going deep into for S researchs, it is found that some internal histocytes can generate H2S, it be after nitric oxide (nitric oxide,
NO the third endogenous gas signaling molecule) and after carbon monoxide (carbon monoxide, CO) being found.Increasingly
Many experiments show, H2S is not only a kind of endogenic gaseous signal molecule, and with various kinds of cell protective effect, participates in
Internal a variety of pathophysiological processes.In mammal, using cysteine or cysteine derivative as substrate catalysis generation H2S
Endogenous enzymes there is relative tissue specificity, cystathionie-γ-lyases (cystathionine- γ-lyase, CGL) is main
Expressed in cardiovascular system, cystathionie-beta-synthetase (cystathionine- β-synthase, CBS) is main in maincenter god
Through being expressed in system, liver and pancreatic tissue.Mercaptopyruvate transsulphurase (3-mercaptopyruvate
Sulfurtransferase, 3-MST) and cysteine lyase (cysteine lyase, CL) also be responsible for catalyzing endogenous property
H2S generation.With H2Important function of the S in biosystem is little by little realized, and is differentiated and is detected biological endogenous property H2S's
Method is of increasing concern, H in detection biological sample2The accuracy and reliability of S concentration for understanding that its function is extremely important,
But due to H2S is volatilizable gas molecule, is influenceed in vivo by various complicated factors, causes its detection difficulty high,
Testing result is unstable.And detect the hydrogen sulfide synthetase activity performance in biological tissue or cell and preferably reflect that the histocyte is produced
Raw H2S ability, relative to the H in directly detection organism2S contents, this method is more reliably and stably.So, it is directly perceived, special
It is different, quantitatively the activity of hydrogen sulfide synzyme is particularly important in detection biological tissue or cell.
Traditional detection H2S methods such as colorimetric method, electrochemical methods, gas chromatography and sulphide precipitation are all needed
Complicated sample treatment is wanted, fluorescence detection method is due to simplicity, and sensitivity is high, favorable repeatability, and can realize visualization
Detection, therefore, new luciferase assay reagent and detection method turn into the focus of research.The sensitivity of hydrogen sulfide fluorescence probe is high,
Instruction system can be studied as biological substance, and available for H2Hydrogen sulfide synthase activity in S quantitative detection and histocyte
Indirect determination, be also widely used in the field such as diagnosis of research Microenvironment Properties and disease in addition, fluorescence detection method
It can yet be regarded as a kind of detection means directly perceived, special, easy, be expected to replace traditional detection method.
The method of traditional detection hydrogen sulfide synthase activity has reaction bulb-methylene blue laws, but in this method repeatedly
Freeze-thaw method cracking histocyte step seems particularly cumbersome, and it detects the H in reaction bulb absorption system2S contents are also not so good as this hair
Bright fluorescence imaging method is directly perceived, easy and sensitive.Fluorescence probe employed in the method for the present invention is the new sulphur of phenylpropyl alcohol two
Alcoholic compound, is the lactone conformation of a closing, and stability is good, and use (- 20 DEG C) can be preserved for a long time, no visible region
Absorption characteristic, the faint (fluorescence quantum yield of probe autofluorescence：Φ=0.003), only Fluorescence Increasing (fluorescent quantum after the reaction
Yield：Φ=0.392), signal to noise ratio is high during for fluorescence imaging, and available for the quantitative determination of fluorescence；The fluorescence probe is in phosphorus
Detection in acid buffering solution is limited to micromole (μm ol/L), can detect the 10 micromolar H in blood plasma and cell2S concentration,
Detection results are stable.Fluorescence probe and H2Change is rapid before and after S reactions, and quantum yield is higher, under the conditions of 37 DEG C and H2S reacts
1h is up to highest fluorescence intensity, it is adaptable to H in reaction system2S quick measure；Its is easy to operate, and testing conditions are easily realized,
Favorable repeatability, visualization；, can specifically and H also, fluorescence probe selectivity is high2S reacts, not by other biological mercaptan
As cysteine, glutathione are disturbed.Therefore, this method holds promise as a kind of new hydrogen sulfide synthase activity detection skill
Art is applied.
Three content of the invention
The present invention need solve the problem of be：
1. provide a kind of based on H2S fluorescence probe, the new method available for detection hydrogen sulfide synthase activity.It is this
Fluorescence probe detection enzymatic activity method can be the mechanism during some important zymetologys in research histocyte, illustrate biosystem
In some important signal paths, early diagnosis for some major diseases and prevention provide good supplementary means.
2. H is provided2S fluorescence probes detect the application approach of hydrogen sulfide synthase activity.
The technical scheme is that：
1. a kind of fluorescence probe of use hydrogen sulfide detects the new method of hydrogen sulfide synthase activity：
(1) fluorescence probe employed in method of the invention is the new mercaptan compound of phenylpropyl alcohol two, is a closing
Lactone conformation, the absorption characteristic of no visible region, the faint (fluorescence quantum yield of probe autofluorescence：Φ=0.003), only
Fluorescence Increasing (fluorescence quantum yield after the reaction：Φ=0.392), MW=591.65, its structural formula is：
(2) fluorescence probe and H employed in method of the invention2The principle of luminosity and its specificity of S reactions are original：
The fluorescence probe is the lactone conformation of a closing, the absorption characteristic of no visible region, probe
And H (Nonfluorescent)2S reacts in phosphate buffered saline solution (PBS, pH value 7.4), generates fluorogen
And 3H-1,2- benzene dimercaptopropane -3- ketone (Fluorescent).
H2S is a kind of active nucleophilic agent, and nucleophilic displacement of fluorine can be participated in biosystem, detects H2S selectivity, it is crucial
It is to be distinguished with other biological nucleopilic reagent, especially biological thiol such as cysteine and glutathione.Theoretically,
H2S may be considered the mercaptan do not replaced, and can carry out necleophilic reaction twice, and other mercaptan such as cysteine is an alkane
Base nomercaptan, can only carry out a necleophilic reaction.H2S free sulfhydryl groups (SH) and most electrophilic subdivision A in fluorescence probe structure
Reaction generation intermediate product A1, if another electrophilic daughter is in place, the ester group in such as A1, SH groups can undergo one
The process of individual spontaneous cyclization, release fluorescence and product B.This method not only can stably capture H2S is degradable so as to generate
Product B, can also quantitatively determine fluorescence intensity, it is easy intuitively to reflect H again2S contents.A can also be with biological thiol such as
Cysteine etc. reacts, but generation product A2 is unable to spontaneous cyclization release fluorogen.Therefore, that has reported is new by H2S is situated between
The phenylpropyl alcohol mercaptan structure led, it produces fluorescence signal to H2S has higher selectivity, not by other biological mercaptan such as half Guang ammonia
Acid, glutathione interference.
(3) reaction bulb absorption system of the invention is formulated as follows：
A. 100mmol/L phosphate buffers (PBS, PH=7.4) are prepared
A liquid：1mol/L K2HPO4·3H2O (MW=228.22) takes 9.13g+40ml ddH2O；
B liquid：1mol/L KH2PO4(MW=136.09) 1.36g+10ml ddH are taken2O；
A liquid 40.1ml+B liquid 9.9ml are taken, after mixing, ddH are used2O is settled to 500ml；
B. detection H is prepared2S fluorescence probe liquid：4mg fluorescence probes (MW=591.65) are dissolved in 13.5ml acetonitriles,
Compound concentration 50mmol/L fluorescence probe mother liquor, it is -20 DEG C long-term to preserve, in use, first taking 100mmol/L phosphate buffers
(PBS, PH=7.4) 500 μ l, add the μ l of acetonitrile 500, take the μ l of fluorescence probe mother liquor 20 to be added thereto after mixing and are mixed with,
20 μm of mol/L of compound concentration fluorescence probe working solution；
C. Cetrimide (CTAB) is prepared：Take 364mg CTAB (MW=364.45) to be dissolved in 100ml ethanol to be made into
10mmol/L Cetrimide mother liquor, normal temperature is preserved, in use, using 10mmol/L phosphate buffers (PBS, PH=by 1: 1000
7.4) it is diluted, is made into 10 μm of ol/L of concentration Cetrimide working solution；
D. reaction bulb absorption system is prepared：20 μm of mol/L of the concentration μ l of hydrogen sulfide probe face liquid 100 are taken, it is basic herein
It is upper to add reaction adjuvant Cetrimide mother liquor (CTAB, 10mmol/L) 10 μ l, and be settled to 10ml with phosphate buffer (PBS), i.e.,
The final concentration of 10 μm of ol/L of CTAB, take the reaction system 1ml prepared to add reaction bulb absorption cell, matching while using.
(4) reaction bulb reaction system of the invention is formulated as follows：
A. 100mmol/L phosphate buffers (PBS, PH=7.4) are prepared
A liquid：1mol/L K2HPO4·3H2O (MW=228.22) takes 9.13g+40ml ddH2O；
B liquid：1mol/L KH2PO4(MW=136.09) 1.36g+10ml ddH are taken2O；
A liquid 40.1ml+B liquid 9.9ml are taken, after mixing, ddH are used2O is settled to 500ml；
B. the phosphoric acid buffer that 1mol/L Cys (MW=121.16) take 0.12g+1ml above-mentioned (1) to prepare is prepared
Liquid；
C. prepare 20mol/L (0.5%) PLP (MW=265.1) and take what 0.50g+100ml above-mentioned (1) was prepared
Phosphate buffer；
(5) detection method experimental procedure of the invention is as follows：
A. for tissue sample, first it is cut into small pieces, then grinds, for cell, histiocyte lysate (strong) is cracked
(green skies biotechnology institute, Nantong, China)；
B. histocyte pyrolysis product is through protein quantification；
C. histocyte pyrolysis product is added in the reaction tank of reaction bulb outer ring, while adding 2mmol/L 5- phosphoric acid
The Cys and lysate of pyridoxal and 10mmol/L.This is the reaction system of reaction bulb；
D. absorption system is added in the absorption cell of inner ring, that is, adds a kind of hydrogen sulfide fluorescence probe (20 μm of ol/L) 100 μ l
With reaction adjuvant Cetrimide (CTAB, 10 μm of ol/L) 100 μ l, and 10ml is settled to phosphate buffer (PBS).
E. closed absorbing reaction 60min is carried out under the conditions of 37 DEG C；
F. fluoremetry or fluorescence imaging.
2.H2S fluorescence probes detect the application approach of hydrogen sulfide synthase activity
By the H of the present invention2S fluorescence probes are used for the histiocytic detection of sulfide hydrogen synzyme in biosystem, raw
Thing tissue or Hemapoiesis H2The analysis detection of S function and fluorescence imaging detection.
Compared with prior art, its advantage is the new method that the present invention is detected to hydrogen sulfide synthase activity：
(1) histiocyte lysate cell lysis tissue, lysate does not influence the catalytic activity of enzyme in itself, and it cracks degree
It is more thorough than multigelation method；
(2) probe steady is good, and use (- 20 DEG C) can be preserved for a long time；
(3) easy to operate, testing conditions are easily realized, favorable repeatability, Visual retrieval；
(4) probe selectivity is high, specifically can be reacted with H2S, it, which produces fluorescence signal, unique selectivity to H2S,
Do not disturbed by other biological mercaptan such as cysteine, glutathione.
(5) probe and H2Change is rapid before and after S reactions, and quantum yield is higher, under the conditions of 37 DEG C and H2S reactions 1h is reachable
Highest fluorescence intensity, it is adaptable to H in reaction system2S quick measure；
(6) probe is detected in phosphate buffer solution is limited to micromole (μm ol), detects sensitive；But it can detect in blood
10 micromolar H in slurry and cell2S concentration, Detection results are stable.
(7) probe autofluorescence is faint (Φ=0.003), only Fluorescence Increasing (Φ=0.392) after the reaction, for fluorescence
Relatively low signal to noise ratio will not be caused during imaging, and available for the quantitative determination of fluorescence；
The H of the present invention2S fluorescence probes detection hydrogen sulfide synthase activity can be research H2The signal path that S-phase is closed, and
H2Meanings and diagnosis of the S in some diseases or organism during diease occurrence provide preferable research method.
The H of the present invention2S fluorescence probes detection hydrogen sulfide synthase activity method can intuitively reflect that biological a certain histocyte exists
H is produced under specific physiology or pathology environment2S ability, testing result can be used for fluorescence imaging, have in biotechnology industry
Good practicality.
Four brief description of the drawings
Fig. 1：For reaction bulb figure, its volume is 50ml, and reaction system is added in the reaction tank of reaction bulb outer ring, and absorption system adds
In the absorption cell of inner ring, absorption pool volume is 2ml.
Fig. 2：New absorption system (the H of the adjuvant Cetrimide containing reaction2S fluorescence probes working solution) detection NaHS donors institute
The H of release2S contents, control group adds the PBS of certain volume to reaction tank, and experimental group adds same volume not to reaction tank
With the NaHS donors of concentration, two groups add identical absorption system, 37 DEG C, jog 1h, with reaction bulb-fluorescence all in absorption cell
Sonde method fluorescence intensity, fluorescence intensity can reflect H in absorbing liquid2S content.
Fig. 3：Different soak times, H2The H that S fluorescence probes detection NaHS donors are discharged2S contents, control group is to reaction
Pond adds the PBS of certain volume, and experimental group adds the NaHS donors of the various concentrations of same volume to reaction tank, and two groups are all being inhaled
Identical absorption system (the H of the adjuvant Cetrimide containing reaction is added in receives pond2S fluorescence probes working solution), 37 DEG C, when jog is different
Between, with reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorbing liquid2S content.
Fig. 4：New absorption system (the H of the adjuvant Cetrimide containing reaction2S fluorescence probes working solution) detection NaHS donors institute
The H of release2S contents, prepare the NaHS donors of various concentrations, are directly added into identical H respectively2S fluorescence probes, are swept with all-wave length
Retouch multi-functional readout instrument (Varioskan Flash3001) (maximum excitation wavelength X ex (max)=476nm, maximum emission wavelength λ
Em (max)=513nm).
Fig. 5：Detect H2S fluorescence probes are quenched situation, and control group adds the PBS of certain volume, experimental group to reaction tank
600 μm of ol/L Na of same volume are added to reaction tank2S donors, two groups add identical absorption system all in absorption cell
(the H of the adjuvant Cetrimide containing reaction2S fluorescence probes working solution), 37 DEG C, jog 1h detects fluorescence with reaction bulb-fluorescence probe method
Intensity, in its fluorescent quenching situation of observed under fluorescent light.
Fig. 6：Left-handed-glucose of dextrorotation-glucose (D-Glu) and osmotic pressure control of the HaCaT cells through various concentrations
(L-Glu) after processing 72h, original multigelation method, the histiocyte lysate provided with green skies biotechnology are improved
(strong) is cracked.With the H of the high sugar processing HaCaT cells of reaction bulb-methylene blue development process detection of improvement2S synzyme
(CBS) activity.
Fig. 7：By 50 μm of ol/L S-N H2S donors (Yz-2-54) add in the HaCaT cells of culture and handle 1h, use H2S is visited
Pin is directly detected in culture medium and intracellular H2S release conditions.
Five embodiments：
The present invention is specifically described below by embodiment, but the present invention is not limited by following embodiments.
Embodiment 1：The H that new absorption system detection NaHS donors are discharged2S contents
There is document to show, HS in NaHS solution-/ H2S ratios are 3: 1, so being largely HS in NaHS solution-.Control group
The PBS of certain volume is added to reaction tank, experimental group adds the various concentrations NaHS donors of same volume to reaction tank, and two groups all
Identical absorption system (the H of the adjuvant Cetrimide containing reaction is added in absorption cell2S fluorescence probes working solution), 37 DEG C, jog
1h, with reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorbing liquid2S content.As a result such as Fig. 2
It has been shown that, the fluorescence intensity of high concentration group (600 μm of ol/L) is more than low concentration group (200 μm of ol/L), and experimental group fluorescence intensity is obvious
More than control group.
Embodiment 2：Different soak times, H2The H that S fluorescence probes detection NaHS donors are discharged2S contents
Control group adds the PBS of certain volume to reaction tank, and experimental group adds the various concentrations of same volume to reaction tank
NaHS donors, two groups add the identical absorption system (H of the adjuvant Cetrimide containing reaction all in absorption cell2S fluorescence probe works
Make liquid), 37 DEG C, jog different time, with reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect absorbing liquid
Middle H2S content.As a result such as Fig. 3 is shown, the fluorescence intensity of 600 μm of ol/L NaHS groups is more than 200 μm of ol/L NaHS groups, experiment
Group fluorescence intensity is significantly greater than in control group, experimental group, and identical NaHS concentration, 1h groups fluorescence intensity is slightly larger than 1/2h groups,**P
＜ 0.01 is compared with Con-1/2h groups,##P ＜ 0.01 are compared with Con-1h groups.
Embodiment 3：H2S fluorescence probes directly detect the H that NaHS donors are discharged2S contents
The NaHS donors of various concentrations are prepared respectively, add identical H2S fluorescence probes, are read with full wavelength scanner is multi-functional
Number instrument (Varioskan Flash3001) (maximum excitation wavelength X ex (max)=476nm, maximum emission wavelength λ em (max)=
513nm).As a result such as Fig. 4 is shown, with H2The rise of S concentration, fluorescence intensity is accordingly raised, in dose dependent.**P ＜ 0.01
Compared with Blank groups,##P ＜ 0.01 are compared with 0 μm of ol/L NaHS group.
Embodiment 4：Detect H2Situation is quenched in S fluorescence probes
Control group adds the PBS of certain volume to reaction tank, and experimental group adds 600 μm of ol/L of same volume to reaction tank
NaHS donors, two groups add the identical absorption system (H of the adjuvant Cetrimide containing reaction all in absorption cell2S fluorescence probes work
Liquid), 37 DEG C, jog 1h, with reaction bulb-fluorescence probe method fluorescence intensity, in its fluorescent quenching situation of observed under fluorescent light, knot
Really such as Fig. 5 is shown, H2S fluorescence probes are exposed under fluorescence occurs more apparent be quenched after 3min.
Embodiment 5：Methylene blue detection method determines the CBS activity of high sugar processing HaCaT cells
Left-handed-glucose (L- of dextrorotation-glucose (D-Glu) and osmotic pressure control of the HaCaT cells through various concentrations
Glu) after processing 72h, improved on original reaction bulb-methylene blue development process, the group provided with green skies biotechnology
Cell pyrolysis liquid (strong) is knitted to be cracked.Detect intracellular H2The activity of S synzyme.As a result such as Fig. 6 is shown, high sugar processing can suppress
CBS activity, this effect is unrelated with osmotic pressure.*P ＜ 0.05,**P ＜ 0.01 are compared with 5mmol/L D-Glu groups.Moreover, splitting
Solution liquid does not influence the catalytic activity of enzyme in itself, and cracking degree is more thorough than original multigelation method, and experiment has good stability.
Embodiment 6：H2The new S-N H of S probe in detecting2S donors (Yz-2-54) discharge H2S ability
S-N H2S donors (Yz-2-54) molecular formula such as Fig. 7 (2) is shown.50 μm of ol/L Yz-2-54 are added into culture
Handled in HaCaT cells in 1h, discovery culture medium has H with intracellular2S is generated.As a result such as Fig. 7 (1) displays, new Yz-
2-54 can discharge H2S molecules, and by H2S fluorescence probes are detected.
Claims (4)
1. the method that a kind of fluorescence probe of use hydrogen sulfide detects hydrogen sulfide synthase activity, it is characterized in that：
(1) this detection method is applied to detection CBS, CSE and CL activity, and wherein CL needs to it is possible to additionally incorporate nitrite, real
Test step as follows：For tissue sample, first it is cut into small pieces, then grinds, for cell, uses the green skies biotechnology of China Nantong
The histiocyte lysate provided is cracked；Product of cell lysis is added in reaction bulb, reaction system after protein quantification
It is added in the reaction tank of outer ring, includes 2mmol/L PLP 400 μ l, the PLP MW=used
265.1,10mmol/L Cys 40 μ l, the Cys MW=121.16 used, and histiocyte lysate, and
2ml is settled to 100mmol/L, PH=7.4 phosphate buffer；Absorption system is added in the absorption cell of inner ring, that is, adds 20 μ
Mol/L hydrogen sulfide fluorescence probe working solution 1ml, wherein the reaction adjuvant Cetrimide containing 10 μm of ol/L；In 37 DEG C of closed loop
Absorbing reaction 60min in border, takes the solution of absorption cell to can be used for fluoremetry and fluorescence imaging；
(2) detection hydrogen sulfide synthase activity is tested, and the histiocytic cleavage method in this experiment is：With the green cloud of China Nantong
The histiocyte lysate that its biotechnology is provided is cracked, and the cracking of 100 μ l cells is added for 55mm Tissue Culture Dish
Liquid；
(3) detection hydrogen sulfide synthase activity is tested, and the absorption system of this experiment is：Reaction adjuvant bromine alkane containing 10 μm of ol/L
The hydrogen sulfide fluorescence probe working solution of ammonium, the absorbing reaction 60min in 37 DEG C of closed environment；
(4) detection hydrogen sulfide synthase activity is tested, and the detection method in this experiment is：Absorption cell solution is taken, it is glimmering with U.S. AMG
Light microscope carries out fluorescence imaging, or with model Varioskan Flash3001, maximum excitation wavelength X ex=476nm, most
The big emission wavelength lambda em=513nm multi-functional readout instrument of full wavelength scanner carries out fluoremetry, draws H2S standard curves can enter
Row quantitative analysis.
2. the reaction of the method for the fluorescence probe detection hydrogen sulfide synthase activity of a kind of use hydrogen sulfide according to claim 1
The compound method of bottle absorption system, it is characterized in that：
(1) 100mmol/L, PH=7.4 phosphate buffer are prepared
A liquid：1mol/L K2HPO43H2O MW=228.22 take 9.13g+40ml ddH2O；
B liquid：1mol/L KH2PO4 MW=136.09 take 1.36g+10ml ddH2O；
A liquid 40.1ml+B liquid 9.9ml are taken, after mixing, 500ml is settled to ddH2O；
(2) detection H is prepared2S fluorescence probe liquid：4mg, MW=591.65 fluorescence probe are dissolved in 13.5ml acetonitriles, prepared
Concentration 50mmol/L fluorescence probe mother liquor, it is -20 DEG C long-term to preserve, in use, first taking 100mmol/L, PH=7.4 phosphoric acid to delay
The μ l of fliud flushing 500, add the μ l of acetonitrile 500, take the μ l of fluorescence probe mother liquor 20 to be added thereto after mixing and are mixed with, compound concentration 20
μm mol/L fluorescence probe working solution；
(3) Cetrimide is prepared：364mg, MW=364.45 Cetrimide is taken to be dissolved in the bromine for being made into 10mmol/L in 100ml ethanol
Alkane ammonium mother liquor, normal temperature is preserved, in use, being diluted by 1: 1000 with 10mmol/L, PH=7.4 phosphate buffer, is made into
10 μm of ol/L of concentration Cetrimide working solution；
(4) reaction bulb absorption system is prepared：20 μm of mol/L of the concentration μ l of hydrogen sulfide probe face liquid 100 are taken, are added on this basis
Enter the 10mmol/L μ l of reaction adjuvant Cetrimide mother liquor 10, and be settled to 10mmol/L, PH=7.4 phosphate buffer
10ml, i.e. Cetrimide final concentration of 10 μm of ol/L, take the reaction system 1ml prepared to add reaction bulb absorption cell, now with existing
Match somebody with somebody.
3. the reaction of the method for the fluorescence probe detection hydrogen sulfide synthase activity of a kind of use hydrogen sulfide according to claim 1
The compound method of bottle reaction system, it is characterized in that：
(1) 100mmol/L, PH=7.4 phosphate buffer are prepared
A liquid：The K2HPO43H2OMW=228.22 that 1mol/L K2HPO43H2O are used takes 9.13g+40ml ddH2O；
B liquid：The KH2PO4MW=136.09 that 1mol/L KH2PO4 are used takes 1.36g+10ml ddH2O；
A liquid 40.1ml+B liquid 9.9ml are taken, after mixing, 500ml is settled to ddH2O；
(2) 1mol/L Cys are prepared, the Cys MW=121.16 used takes 0.12g+1ml above-mentioned (1) to match somebody with somebody
The phosphate buffer of system；
(3) 20mol/L PLP is prepared, the PLP MW=265.1 used is taken on 0.50g+100ml
State the phosphate buffer of (1) preparation.
4. a kind of method of the fluorescence probe detection hydrogen sulfide synthase activity of use hydrogen sulfide is in biology according to claim 1
Application in engineering industry.
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