CS269204B1 - Method of insoluble labelled substrates preparation for proteolytic activity determination - Google Patents
Method of insoluble labelled substrates preparation for proteolytic activity determination Download PDFInfo
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- CS269204B1 CS269204B1 CS881123A CS112388A CS269204B1 CS 269204 B1 CS269204 B1 CS 269204B1 CS 881123 A CS881123 A CS 881123A CS 112388 A CS112388 A CS 112388A CS 269204 B1 CS269204 B1 CS 269204B1
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- CS
- Czechoslovakia
- Prior art keywords
- insoluble
- proteolytic activity
- labeled
- activity determination
- labelled substrates
- Prior art date
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- 239000000758 substrate Substances 0.000 title claims abstract description 12
- 230000002797 proteolythic effect Effects 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 title description 3
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 229920002401 polyacrylamide Polymers 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 3
- 238000006116 polymerization reaction Methods 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 10
- 102000035195 Peptidases Human genes 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical class NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000016942 Elastin Human genes 0.000 description 2
- 108010014258 Elastin Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229920002549 elastin Polymers 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- 239000000985 reactive dye Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WRXNJTBODVGDRY-UHFFFAOYSA-N 2-pyrrolidin-1-ylethanamine Chemical compound NCCN1CCCC1 WRXNJTBODVGDRY-UHFFFAOYSA-N 0.000 description 1
- IHDBZCJYSHDCKF-UHFFFAOYSA-N 4,6-dichlorotriazine Chemical compound ClC1=CC(Cl)=NN=N1 IHDBZCJYSHDCKF-UHFFFAOYSA-N 0.000 description 1
- ORLGPUVJERIKLW-UHFFFAOYSA-N 5-chlorotriazine Chemical compound ClC1=CN=NN=C1 ORLGPUVJERIKLW-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- KUIXZSYWBHSYCN-UHFFFAOYSA-L remazol brilliant blue r Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(N)=C2C(=O)C3=CC=CC=C3C(=O)C2=C1NC1=CC=CC(S(=O)(=O)CCOS([O-])(=O)=O)=C1 KUIXZSYWBHSYCN-UHFFFAOYSA-L 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Příprava nerozpustných značených substrátů pro stanovení proteolytické aktivity podle řeSení se provádí zapolymerováním značených bílkovin do struktury polyakrylamidu.Preparation of insoluble labeled substrates for proteolytic determination the activity according to the invention is carried out by polymerization labeled protein to polyacrylamide structure.
Description
CS 269204 BlCS 269204 Bl
Vynález se týká způsobu přípravy nerozpustných značených substrátů pro stanovení proteolytické aktivity.The present invention relates to a process for preparing insoluble labeled substrates for the determination of proteolytic activity.
Pro stanovení aktivity proteolytických enzymů je možno použít celou řadu substrá-tů. Mezi nimi významná místo zaujímají nerozpustná bílkovinná značená substráty. Mezine’ patří například kožní práSek s navázanou Remazolovou brilantní modří, elastin s na-vázanou kongo červení, fibrin obarvený indigokaraínem, želatina zeeítíná glutaraldehy-dem v přítomnosti nigrosinu, želatina značená fluoresceinem a navázaná na Sepharosu 4B,kolagen značený nebo sítovaná bílkoviny s kovalentně navázaným reaktivním barvivém.A variety of substrates can be used to determine the activity of proteolytic enzymes. Among them, the insoluble protein-labeled substrates occupy an important place. The mesine includes, for example, a powder powder with bound Remazol brilliant blue, elastin with bound congo red, fibrin stained with indigocaraine, gelatin crosslinks with glutaraldehyde in the presence of nigrosin, gelatin labeled with fluorescein and bound to Sepharose 4B, collagen labeled or crosslinked proteins with covalently bound reactive dye.
Dosud používaná způsoby přípravy nerozpustných značených bílkovinných substrátůpro stanovení proteolytická aktivity spočívají v tom, že se na přirozenou nerozpust-nou bílkovinu (například kolagen, keratin, elastin, fibrin) kovalentnS nebo adsorbcínaváže vhodná značka, nebo se běžná rozpustná bílkoviny v roztoku zesítí bifunkčnímčinidlem, přičemž se vytvoří nerozpustná sítovaná bílkoviny, na která se v druhámStupni kovalentnS nebo absorbcí naváže vhodná značka. DalSÍ možností je, že se v roz-toku bílkoviny suspenduje nebo rozpustí vhodná značka a potom se provede zesítSní bíl-koviny vhodným bifunkčním činidlem; značka je zabudována do struktury nerozpustná bíl-koviny. Je taká možná značenou rozpustnou bílkovinu navázat na vhodnou matrici. Deri-váty kaseinu je možná tepelným zásahem převást na nerozpustnou formu.The methods used to prepare insoluble labeled proteinaceous substrates for the determination of proteolytic activity have hitherto been such that a suitable label is applied to the natural insoluble protein (e.g., collagen, keratin, elastin, fibrin), or the conventional soluble protein in the solution is crosslinked with a bifunctional agent, to form insoluble crosslinked proteins to which a suitable label binds in the second stage or by absorption. Another possibility is that a suitable label is suspended or dissolved in the protein solution and then the crosslinking protein is carried out with a suitable bifunctional agent; the label is incorporated into the insoluble protein structure. It is also possible to bind the labeled soluble protein to a suitable matrix. The casein derivatives may be converted to an insoluble form by heat treatment.
Způsob přípravy nerozpustných značených substrátů pro stanovení proteolytickáaktivity podle vynálezu spočívá v tom, že se značená bílkovina zabuduje do strukturypolyakrylamldu. Vzniklý produkt se převede na jemná částice, která se důkladně promy-jí vodou a organickými rozpouštědly a potom se usuSÍ. Působením proteolytických enzy-mů se ze zabudovaná bílkoviny odStěpují značená peptidy, jejichž množství je úměrnáaktivitě proteinasy, a je možná je stanovit například spektrofotometricky, fluorlmet-ricky nebo radiometricky, po odfiltrování nebo odstředění nerozloženého nerozpustnéhosubstrátu.The process of preparing insoluble labeled substrates for the determination of proteolytic activity according to the invention consists in incorporating the labeled protein into the polyacrylamide structure. The resulting product is converted into fine particles which are thoroughly washed with water and organic solvents and then dried. The action of proteolytic enzymes involves the incorporation of labeled peptides by the incorporated proteins, the amount of which is proportional to the activity of the proteinase, and may be determined, for example, spectrophotometrically, fluorometrically or radiometrically, after filtering off or centrifuging the undissolved insoluble substrate.
Vynález je dokumentován příklady. Příklad 1The invention is illustrated by examples. Example 1
Na kasein se kovalentnS naváže reaktivní barvivo dichlorotriazinového nebo mono-chlorotriazinového typu (například 0sta2inová červeň S-53, Ostazinová modř S-2G neboOstazinová modř H-5R) podle standardního postupu. 5 g značeného kaseinu se rozpustív 70 ml 10% Na2HPO^.12 HjO. Po rozpuštění se přidá 28,8 g akrylamidu a 1,2 g N,N'--methylen-bis-akrylamidu. Po rozpuětění se objem roztoku upraví na 100 ml, roztokse odvzduění a přidá se 1,5 ml 10% persíranu amonného a 150 ul N,N,N' ,N'-tetramet-hylenethylendiaminu (TEMED) . Po promíchání se směs ponechá polymerovat. Po 24 hodi-nách se vzniklý gel rozkrájí na malá kousky a homogenizuje se v nožovém mixeru. Jem-ná částice gelu se zabudovanou značenou bílkovinou se na fritě promyjí důkladně vo-dou a etanolem a usuěí se při laboratorní teplotě. Příklad 2The reactive dye of the dichlorotriazine or mono-chlorotriazine type (e.g. 0sta2in red S-53, Ostazin blue S-2G or Ostazin blue H-5R) is coupled to casein by standard procedure. 5 g of labeled casein is dissolved in 70 ml of 10% Na2HPO4 · 12 H2O. After dissolution, 28.8 g of acrylamide and 1.2 g of N, N '-methylene-bis-acrylamide are added. After dissolution, the volume of the solution is adjusted to 100 ml, the solution is vented and 1.5 ml of 10% ammonium persulfate and 150 µl of N, N, N ', N'-tetramethylene ethylenediamine (TEMED) are added. After mixing, the mixture is allowed to polymerize. After 24 hours, the resulting gel is cut into small pieces and homogenized in a knife mixer. The fine gel particles with embedded labeled protein are washed thoroughly on the frit with water and ethanol and dried at room temperature. Example 2
Do zkumavek se naváží po 70 mg substrátu, připraveného podle bodu 1. Přidají se3 ml vhodného pufru a substrát 3e ponechá bobtnat při pracovní teplotě po dobu 20min. Potom se do zkumavek přidá po 1 ml roztoku obsahujícího proteinasy a směs aeprotřepe. Po vhodně dlouhé Inkubaci (například 30 min při 37 až 50 °C) se substrátodfiltruje nebo odstředí a filtrát nebo supernatant se proměří při vhodné vlnovédélce (520 nm pro Ostazinovou červeň S-5B, 590 nm pro Ostazinovou modř H-5R nebo620 nm pro Ostazinovou modř S-20). Trypsin bylo možno dspěěně stanovit v rozsahukoncentrací 0,1 až 10 ug/ ml.Weigh 70 mg of the substrate prepared according to step 1 into the tubes. Add 3 ml of the appropriate buffer and allow substrate 3e to swell at room temperature for 20 min. Thereafter, 1 ml of a solution containing proteinases and a shake mixture is added to the tubes. After a suitably long incubation (for example, 30 min at 37 to 50 ° C), the substrate is filtered or centrifuged and the filtrate or supernatant is measured at an appropriate wavelength (520 nm for Ostazine red S-5B, 590 nm for Ostazin blue H-5R or 620 nm for Ostazin blue S-20). Trypsin was successfully determined in a concentration range of 0.1 to 10 µg / ml.
Claims (1)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS881123A CS269204B1 (en) | 1988-02-23 | 1988-02-23 | Method of insoluble labelled substrates preparation for proteolytic activity determination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS881123A CS269204B1 (en) | 1988-02-23 | 1988-02-23 | Method of insoluble labelled substrates preparation for proteolytic activity determination |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CS112388A1 CS112388A1 (en) | 1989-09-12 |
| CS269204B1 true CS269204B1 (en) | 1990-04-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS881123A CS269204B1 (en) | 1988-02-23 | 1988-02-23 | Method of insoluble labelled substrates preparation for proteolytic activity determination |
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| Country | Link |
|---|---|
| CS (1) | CS269204B1 (en) |
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1988
- 1988-02-23 CS CS881123A patent/CS269204B1/en unknown
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| Publication number | Publication date |
|---|---|
| CS112388A1 (en) | 1989-09-12 |
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