CN102809541B - Determination method of enzyme activity of collagenase - Google Patents
Determination method of enzyme activity of collagenase Download PDFInfo
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- 238000000034 method Methods 0.000 title abstract description 5
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a determination method of enzyme activity of collagenase, and aims to solve the problems of non-uniform enzyme activity detection standard and poor data consistency existing in the prior art. The determination method comprises the following steps of: (1) preparing a reagent; (2) preparing hydroxyproline gradient samples; (3) determining the hydroxyproline gradient samples, drawing a C-A standard curve according to a determined absorbance A and hydroxyproline gradient sample concentration C and fitting a standard curve formula; (4) preparing enzymatic hydrolysate to be determined: adding insoluble collagen or gelatin serving as a collagen substrate into an enzyme solution to be determined for enzymatic hydrolysis, centrifuging to obtain supernatant, and diluting the supernatant to prepare the enzymatic hydrolysate to be determined; (5) preparing a blank solution; (6) determining the enzyme activity and determining the absorbance A of the enzymatic hydrolysate to be determined according to the operation conditions of the step (3); (7) calculating the hydroxyproline concentration in the enzymatic hydrolysate to be determined; and (8) calculating the enzyme activity of the collagenase. The determination method has the characteristics of uniform enzyme activity detection standard, capability of solving the problem of substrate difference, high measuring accuracy and consistent enzyme activity data.
Description
Technical field
The present invention relates to a kind of proteinase activity assay method, particularly relate to the assay method that a kind of Collagenase enzyme is lived.
Background technology
Collagen (Collagen) is the boiomacromolecule of a kind of relative molecular weight more than 300,000 of being synthesized by zooblast, extensively be present in animal skeleton, tendon, cartilage, skin and other connective tissue, account for 1/3 of mammal total protein, in bone and flesh key total protein more than 90%, in skin total protein more than 50%, collagen has unique triple-helix structure, is difficult to by general protease hydrolytic, can only by Collagenase specific for hydrolysis.Collagenase (collagenase), it is a kind of proteolytic enzyme being feature with insoluble hydrolysate collagen albumen, at multiple protease family, as all having existence in Metalloproteinase familv and serine stretch protein enzyme family, at present, Collagenase has been widely used in the field such as medicine, food: in commercial development, be applied to collagen group disassemble to obtain collagen polypeptide; The protrusion of the intervertebral disc etc. is used for the treatment of in medical research; Being separated of cell and organ is applied to as toolenzyme in laboratory study; The research of collagen structure is applied in fundamental research.
Lee's old (" assay method of collagenase activity ", Chinese leather, 2008,37 (11): 24-25) etc. people reviews collagenase activity assay method: using casein as substrate, reaction system is: 0.1mL enzyme liquid, 1mL0.6% casein solution, 50mmol/L pH7.5 Tris-HC1,4mmol/L CaC1
2, 30 DEG C of water-bath 10min, then use the trichloroacetic acid cessation reaction of 10%, and be hydrolyzed the acidity peptide discharged and measure light absorption value under 273nm, enzyme activity is defined as: it is an enzyme activity unit that every milligram of albumen generation per minute is equivalent to equivalent tyrosine micromole number.Prior art Problems existing is: the standard disunity of enzyme definition alive, there is substrate otherness, enzyme live data is inconsistent, and measuring accuracy is not high, thus have impact on application and the popularization of Collagenase and Related product thereof.
Summary of the invention
The present invention is Collagenase Enzyme activity assay standard disunity in order to overcome prior art, there is substrate otherness, deficiency that enzyme live data is inconsistent, provide a kind of standard unified, overcome substrate otherness, assay method that enzyme live data is consistent, measuring accuracy is high Collagenase enzyme is lived.
The present invention is by the following technical solutions:
The assay method that a kind of Collagenase enzyme of the present invention is lived, comprises the steps:
1. reagent prepares: prepare the alcoholic solution that concentration is the TrisCl damping fluid of 0.1mol/LpH7.5, the solution of trichloroacetic acid of volume fraction 10%, the o-phthalaldehyde(OPA) solution of 0.6 ~ 0.8mol/L, the hydroxyproline standard liquid of 0.3mmol/L, the acetate buffer of 2mol/L, triketohydrindene hydrate nitrite ion, volume fraction 60%;
2. hydroxyproline sample preparation: pipette respectively hydroxyproline standard liquid in right amount in color comparison tube dilution be settled to same volume, make some hydroxyproline samples that concentration increases progressively at 0.03 ~ 0.3 μm of ol/mL, to get with the distilled water of hydroxyproline sample same volume in color comparison tube as with reference to sample;
3. hydroxyproline sample determination: add the acetate buffer equal with hydroxyproline sample volume and triketohydrindene hydrate nitrite ion respectively to reference in sample and each hydroxyproline sample, abundant mixing, in 100 ~ 105 DEG C of heating water bath 15 ~ 20min after sealing, cooling, place 10min, then the alcoholic solution dilution of hydroxyproline sample volume 3 times is added, with reference sample for reference solution, the light absorption value of each hydroxyproline sample is surveyed at 520nm place, make C-A typical curve according to the concentration C of hydroxyproline sample and light absorption value A, and simulate typical curve formula;
4. enzymolysis liquid preparation to be measured: take 0.8 ~ 1.2mg collagen substrate, add 0.4 ~ 0.6mlTrisCl damping fluid, in 35 ~ 40 DEG C of preheating 8 ~ 12min, add 0.1ml enzyme liquid to be measured, described enzyme liquid to be measured is long-pending is designated as V
2in 35 ~ 40 DEG C of enzyme digestion reaction Tmin, the value of described T is 25 ~ 35, then add 0.5 ~ 0.7ml solution of trichloroacetic acid to shake up, then the o-phthalaldehyde(OPA) solution adding 0.5 ~ 0.7ml shakes up, and finally adds 0.4 ~ 0.6g silica gel, stirs evenly rear standing 5 ~ 8min, centrifugal supernatant, this supernatant volume is designated as V
1, as enzymolysis liquid to be measured after described supernatant dilution N times;
5. Blank solution: take 0.8 ~ 1.2mg collagen substrate, add 0.4 ~ 0.6ml TrisCl damping fluid, in 35 ~ 40 DEG C of preheating 8 ~ 12min, add 0.5 ~ 0.7ml solution of trichloroacetic acid to shake up, then 0.1ml enzyme liquid to be measured is added, in 35 ~ 40 DEG C of enzyme digestion reaction 25 ~ 35min, the o-phthalaldehyde(OPA) solution adding 0.5 ~ 0.7ml again shakes up, finally add 0.4 ~ 0.6g silica gel, stir evenly rear standing 5 ~ 8min, centrifugal supernatant, described supernatant dilution N doubly after as blank solution, wherein value and the step of N 4. in N value identical;
6. the mensuration of enzyme activity: get enzymolysis liquid to be measured and each certain volume of blank solution in color comparison tube, described volume is designated as V
3, add V respectively
3the acetate buffer of volume and triketohydrindene hydrate nitrite ion, fully mix, and in 100 ~ 105 DEG C of heating water bath 15 ~ 20min after sealing, cooling, places 10min, then add 3 times of V
3the alcoholic solution dilution of volume, is reference solution with blank solution, surveys the light absorption value of enzymolysis liquid to be measured at 520nm place;
7., after measuring enzymolysis liquid light absorption value A to be measured, the Hydroxyproline concentration C in corresponding enzymolysis liquid to be measured in the typical curve formula that 3. substitution step obtains, is obtained;
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein,
cfor the concentration of hydroxyproline in enzymolysis liquid to be measured;
v 1 for supernatant volume;
mWfor hydroxyproline molecular weight;
nfor supernatant extension rate;
v 2 for enzyme liquid to be measured amasss;
tfor the enzyme digestion reaction time.
In the present invention, Collagenase enzyme is lived and is defined as: at 35 ~ 40 DEG C, under pH7.5 condition, it is 1 enzyme activity unit (U) that every milliliter of enzyme liquid Hydrolyzed Collagen per minute produces the enzyme amount being equivalent to 1ug hydroxyproline, and in the present invention, enzyme liquid to be measured refers to that the concentration be mixed with by finished product Collagenase is the enzyme liquid of 0.2mg/L.
Principle of the present invention is as follows: in acid condition, hydroxyproline and ninhydrin reaction generate stable red condensation product, this condensation product has maximum absorption band at wavelength 520nm place, within the specific limits, the concentration of hydroxyproline is directly proportional to its light absorption value, hydroxyproline is the specific amino acid in collagen, stable content, be that the Collagenase enzyme work that standard records overcomes substrate otherness with hydroxyproline, other amino acid generation derivatization reaction beyond the hydroxyproline in o-phthalaldehyde(OPA) and enzymolysis liquid to be measured is adopted in the present invention, derivatization reaction thing adopts silica gel adsorption, hydroxyproline is only had in centrifuged supernatant, eliminate other amino acid whose interference, measuring accuracy is high.
Enzymolysis supernatant dilution N is doubly that 0.03 ~ 0.3 μm of ol/mL refers to that the light absorption value A measured brings the Hydroxyproline concentration that typical curve formula obtains into and controls in 0.03 ~ 0.3 μm of ol/mL to Hydroxyproline concentration, time not in the scope of 0.03 ~ 0.3 μm of ol/mL, adjustment extension rate N, to make the Hydroxyproline concentration recorded control within the scope of this, improve measuring accuracy.
As preferably, step 2. described in refer to 0.1 ~ 1.0ml in right amount, described same volume refers to 1ml, and described concentration is the quantity of the hydroxyproline sample of 0.03 ~ 0.3 μm of ol/mL is 6,7 or 8.
As preferably, the progressive concentration of described 6 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.18,0.24,0.30 μm of ol/mL; The progressive concentration of described 7 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.15,0.18,0.24,0.30 μm of ol/mL; The progressive concentration of described 8 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.15,0.18,0.21,0.24,0.30 μm of ol/mL.
As preferably, described alcoholic solution is ethanolic solution or aqueous isopropanol.Use ethanol or isopropyl alcohol with low cost, security is high.
As preferably, described collagen substrate is insoluble collagen albumen or gelatin.
As preferably, described silica gel is Kiselgel A.Specific surface area is large, and absorption property is good, and chemical stability is good, uses and can meet adsorption entails on a small quantity.
As preferably, step 4. in supernatant be diluted to concentration 0.12 ~ 0.24 μm of ol/mL as enzymolysis liquid to be measured.The scope of Hydroxyproline concentration C is when 0.12 ~ 0.24 μm of ol/mL, and in typical curve, light absorption value A and concentration C are in best linear relationship, and control within the scope of this by the concentration of liquid to be measured for sample, measuring accuracy is the highest.
Therefore, advantage of the present invention is: (1) hydroxyproline is specific amino acid in collagen, stable content, take hydroxyproline as unified standard, overcomes the substrate otherness of Collagenase enzyme activity determination; (2) use o-phthalaldehyde(OPA) as screening agent and silica gel as adsorbent, eliminate other amino acid whose interference, estimating precision is high; (3) required instrument is simple, easy to operate.
Accompanying drawing explanation
Fig. 1 is C-A canonical plotting of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Each reagent be all analyze pure, the reagent solution prepared is applicable to following each embodiment, and unless specified otherwise, solvent is distilled water, each embodiment instrument and material as follows:
(1) spectrophotometer (UV2000)
(2) insoluble collagen albumen: thread collagen (Wo Xindun chemical company, WBC)
(3) gelatin
(4) hydroxyproline, i.e. trans-4-hydroxy-l-proline (boston, u.s.a biotech company, BBI)
(5) silica gel
Embodiment 1
1. reagent prepares
(1) 0.1mol/L TrisCl damping fluid is (containing 50mmol CaCl
2, pH7.5): take 3.15g TrisCl and be dissolved in 200mL distilled water, add 1.11g lime chloride, after fully dissolving, regulate pH to 7.5 with 3mol/L NaOH;
(2) 10% trichloroacetic acid (TCA) solution: 50mL trichloroacetic acid and 450mL distilled water mix;
(3) the o-phthalaldehyde(OPA) distilled water of 0.6mol/L o-phthalaldehyde(OPA) solution: 0.6mol is settled to 1L;
(4) 0.3mmol/L hydroxyproline standard liquid: the hydroxyproline taking 39.4mg is dissolved in distilled water, is settled to 1L;
(5) 2mol/L acetate buffer: the sodium acetate distilled water of 2mol is dissolved, is settled to 1L, the acetic acid distilled water of 2mol is settled to 1L, and then by above-mentioned sodium acetate solution and acetic acid solution 43:7 mixing by volume, pH is 5.4;
(6) triketohydrindene hydrate nitrite ion: get 85mg ninhydrin and the mixing of 15mg hydrindantin, then dissolve by 10ml ethylene glycol monomethyl ether, 4 DEG C keep in Dark Place, described hydrindantin preparation method is as follows: take 0.5g triketohydrindene hydrate, with 12.5ml boil distilled water dissolve, obtain yellow solution, 0.5g vitamin C 25ml temperature distilled water is dissolved, then while stirring vitamin c solution is added drop-wise in ninhydrin solution, continuous appearance precipitation, dropwise rear continuation and stir 15min, then in refrigerator, 4 DEG C are cooled to, filter, precipitation cold water washing 3 times, vacuum drying obtains hydrindantin,
(7) 60% ethanol or isopropyl alcohols: 300mL ethanol or isopropyl alcohol are dissolved in 200mL distilled water.
2. hydroxyproline sample preparation: pipette 0.1 respectively, 0.2,0.4,0.6,0.8, the hydroxyproline standard liquid of 1.0mL is in 25ml color comparison tube, 1mL is settled to distilled water polishing, be diluted to 6 hydroxyproline samples that concentration is respectively 0.03,0.06,0.12,0.18,0.24,0.30 μm of ol/mL, get 1.0ml distilled water in 25ml color comparison tube as reference sample;
3. hydroxyproline sample determination: the acetate buffer and the 1mL triketohydrindene hydrate nitrite ion that add 1mL 2mol/L in reference sample and each hydroxyproline sample respectively, abundant mixing, in 100 DEG C of heating water bath 20min after sealing, cooling, place 10min, add the dilution of 3mL 60% ethanolic solution, with reference sample for reference solution, survey the light absorption value of each hydroxyproline sample at 520nm place, the concentration C of each hydroxyproline sample with the light absorption value A measured in table 1:
Make C-A typical curve according to the concentration C of hydroxyproline sample and light absorption value A, and simulate typical curve formula: C=2.748A-0.2325.
4. enzymolysis liquid preparation to be measured: take 0.8mg insoluble collagen albumen, add the TrisCl damping fluid of 0.4mL 0.1mol/L in 35 DEG C of preheating 8min, add 0.1mL enzyme liquid again, in 35 DEG C of reaction 25min, add the solution of trichloroacetic acid of 0.5mL 10%, abundant mixing is with cessation reaction, then the o-phthalaldehyde(OPA) solution of 0.5mL0.8mol/L is added, shake up reaction 5min, then 0.4g silica gel is added, shake up rear standing 5min, centrifugal supernatant 1.5ml, dilutes 25 times as enzymolysis liquid to be measured using supernatant;
5. Blank solution: take 0.8mg insoluble collagen albumen, add the TrisCl damping fluid of 0.4mL 0.1mol/L, 35 DEG C of preheating 8min, add the trichloroacetic acid of 0.5mL 10%, 0.1mL enzyme liquid is added again after shaking up, 35 DEG C of reaction 25min, then add the o-phthalaldehyde(OPA) solution of 0.5mL0.8mol/L, shake up reaction 5min, then 0.4g silica gel is added, shake up rear standing 5min, centrifugal supernatant 1.5ml, dilutes 25 times as blank solution using centrifugal clear liquid;
6. enzyme activity determination: respectively get 1.0mL enzymolysis liquid to be measured and blank solution; add acetate buffer and the 1.0mL triketohydrindene hydrate nitrite ion of 1.0mL 2mol/L respectively; abundant mixing; in 100 DEG C of heating water bath 20min after sealing; cooling, places 10min, adds the dilution of 3mL 60% ethanol; be reference with blank solution, measuring the light absorption value of enzymolysis liquid to be measured at 520nm place is 0.130;
7. light absorption value 0.130 being substituted into typical curve formula C=2.748A-0.2325, to obtain Hydroxyproline concentration in enzymolysis liquid to be measured be 0.1247 μm of ol/mL.
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.1247 μm of ol/mL; V1=1.5ml; V2=0.1ml; MW=131.3; N=25; T=25min.
Substitute into formula:
=
=246.2
Embodiment 2
1. reagent prepares: with embodiment 1, difference is O-phthalic aldehyde concentration is 0.8mol/l;
2. hydroxyproline sample preparation: pipette 0.1 respectively, 0.2,0.4,0.5,0.6,0.8, the hydroxyproline standard liquid of 1.0mL is in 25ml color comparison tube, 1mL is settled to distilled water polishing, be diluted to 7 hydroxyproline samples that concentration is respectively 0.03,0.06,0.12,0.15,0.18,0.24,0.30 μm of ol/mL, get 1.0ml distilled water in 25ml color comparison tube as reference sample;
3. hydroxyproline sample determination: operating conditions is with embodiment 1, and difference is: bath temperature is 105 DEG C, and the time is 15min, simulates typical curve formula: C=2.7483A-0.2328.
4. enzymolysis liquid preparation to be measured: take 1.2mg insoluble collagen albumen, add the TrisCl damping fluid of 0.6mL 0.1mol/L in 40 DEG C of preheating 12min, add 0.1mL enzyme liquid again, in 40 DEG C of reaction 35min, add the solution of trichloroacetic acid of 0.7mL 10%, abundant mixing is with cessation reaction, then the o-phthalaldehyde(OPA) solution of 0.7mL0.8mol/L is added, shake up reaction 8min, then 0.6g silica gel is added, shake up rear standing 8min, centrifugal supernatant 2.1ml, dilutes 20 times as enzymolysis liquid to be measured using supernatant;
5. Blank solution: take 1.2mg insoluble collagen albumen, add the TrisCl damping fluid of 0.6mL 0.1mol/L, 40 DEG C of preheating 12min, add the trichloroacetic acid of 0.7mL 10%, 0.1mL enzyme liquid is added again after shaking up, 40 DEG C of reaction 35min, then add the o-phthalaldehyde(OPA) solution of 0.7mL0.8mol/L, shake up reaction 8min, then 0.6g silica gel is added, shake up rear standing 8min, centrifugal supernatant 2.1ml, dilutes 20 times as blank solution using centrifugal clear liquid;
6. enzyme activity determination: respectively get 1.0mL enzymolysis liquid to be measured and blank solution; add acetate buffer and the 1.0mL triketohydrindene hydrate nitrite ion of 1.0mL 2mol/L respectively; abundant mixing; in 105 DEG C of water-bath 15min after sealing; cooling, places 10min, adds 3mL 60% isopropanol; be reference with blank solution, measuring the light absorption value of enzymolysis liquid to be measured at 520nm place is 0.142;
7. light absorption value 0.142 being substituted into typical curve formula C=2.7483A-0.2328, to obtain Hydroxyproline concentration in enzymolysis liquid to be measured be 0.158 μm of ol/mL.
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.158 μm of ol/mL; V1=2.1ml; V2=0.1ml; MW=131.3; N=20; T=35min.
Substitute into formula:
=
=248.9
Embodiment 3
1. reagent prepares: with embodiment 1, difference is O-phthalic aldehyde concentration is 0.7mol/l;
2. hydroxyproline sample preparation: pipette 0.1 respectively, 0.2,0.3,0.4,0.6,0.7,0.8, the hydroxyproline standard liquid of 1.0mL is in 25ml color comparison tube, 1mL is settled to distilled water polishing, be diluted to 8 hydroxyproline samples that concentration is respectively 0.03,0.06,0.12,0.15,0.18,0.21,0.24,0.30 μm of ol/mL, get 1.0ml distilled water in 25ml color comparison tube as reference product;
3. hydroxyproline sample determination: operating conditions is with embodiment 1, and difference is: bath temperature is 102 DEG C, the time is 18min; Simulate typical curve formula: C=2.7488A-0.2432.
4. enzymolysis liquid preparation to be measured: take 1.0mg insoluble collagen albumen, add the TrisCl damping fluid of 0.5mL 0.1mol/L in 37 DEG C of preheating 10min, add 0.1mL enzyme liquid again, in 37 DEG C of reaction 30min, add the solution of trichloroacetic acid of 0.6mL 10%, abundant mixing is with cessation reaction, then the o-phthalaldehyde(OPA) solution of 0.6mL0.8mol/L is added, shake up reaction 6min, then 0.5g silica gel is added, shake up rear standing 6min, centrifugal supernatant 1.8ml, dilutes 25 times as enzymolysis liquid to be measured using supernatant;
5. Blank solution: take 1.0mg insoluble collagen albumen, add the TrisCl damping fluid of 0.5mL 0.1mol/L, 37 DEG C of preheating 10min, add the trichloroacetic acid of 0.6mL 10%, 0.1mL enzyme liquid is added again after shaking up, 37 DEG C of reaction 30min, then add the o-phthalaldehyde(OPA) solution of 0.6mL0.8mol/L, shake up reaction 6min, then 0.5g silica gel is added, shake up rear standing 6min, centrifugal supernatant 1.8ml, dilutes 25 times as blank solution using centrifugal clear liquid;
6. enzyme activity determination: respectively get 1.0mL enzymolysis liquid to be measured and blank solution; add the acetate buffer of 1.0mL 2mol/L respectively; abundant mixing, then adds 1.0mL triketohydrindene hydrate nitrite ion respectively, fully mixes; in 102 DEG C of heating water bath 18min after sealing; cooling, places 10min, adds the dilution of 3mL 60% ethanol; be reference with blank solution, measuring the light absorption value of enzymolysis liquid to be measured at 520nm place is 0.135;
7. light absorption value 0.135 being substituted into typical curve formula C=2.7488A-0.2432, to obtain hydroxyproline content in enzymolysis liquid to be measured be 0.128 μm of ol/mL.
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.128 μm of ol/mL; V1=1.8ml; V2=0.1ml; MW=131.3; N=25; T=30min.
Substitute into formula:
=
=251.9
Embodiment 4
1. reagent prepares: with embodiment 1;
2. hydroxyproline sample preparation: with embodiment 1;
3. hydroxyproline sample determination: with embodiment 1;
4. enzymolysis liquid preparation to be measured: take gelatin as collagen substrate, other operating conditions is with embodiment 1;
5. Blank solution: take gelatin as collagen substrate, other operating conditions is with embodiment 1;
6. enzyme activity determination: operating conditions is with embodiment 1, and recording enzymolysis liquid light absorption value A to be measured is 0.129;
7. light absorption value A=0.129 is substituted into typical curve formula C=2.748A-0.2325, obtain Hydroxyproline concentration C=0.122 μm ol/mL in enzymolysis liquid to be measured;
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.122 μm of ol/mL; V1=1.5ml; V2=0.1ml; MW=131.3; N=25; T=25min.
Substitute into formula:
=
=240.3
Embodiment 5
1. reagent prepares: with embodiment 2;
2. hydroxyproline sample preparation: with embodiment 2;
3. hydroxyproline sample determination: with embodiment 2;
4. enzymolysis liquid preparation to be measured: take gelatin as collagen substrate, other operating conditions is with embodiment 2;
5. Blank solution: take gelatin as collagen substrate, other operating conditions is with embodiment 2;
6. enzyme activity determination: operating conditions is with embodiment 2, and recording enzymolysis liquid light absorption value A to be measured is 0.143;
7. light absorption value A=0.143 is substituted into typical curve formula C=2.7483A-0.2328, obtain Hydroxyproline concentration C=0.160 μm ol/mL in enzymolysis liquid to be measured;
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.160 μm of ol/mL; V1=2.1ml; V2=0.1ml; MW=131.3; N=20; T=35min.
Substitute into formula:
=
=251.5
Embodiment 6
1. reagent prepares: with embodiment 3;
2. hydroxyproline sample preparation: with embodiment 3;
3. hydroxyproline sample determination: with embodiment 3;
4. enzymolysis liquid preparation to be measured: take gelatin as collagen substrate, other operating conditions is with embodiment 3;
5. Blank solution: take gelatin as collagen substrate, other operating conditions is with embodiment 3;
6. enzyme activity determination: operating conditions with embodiment 3, recording light absorption value A is 0.131;
7. light absorption value A=0.131 is substituted into typical curve formula C=2.7488A-0.2328, obtain Hydroxyproline concentration C=0.127 μm ol/mL in enzymolysis liquid to be measured;
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein: C=0.127 μm of ol/mL; V1=1.8ml; V2=0.1ml; MW=131.3; N=25; T=30min.
Substitute into formula:
=
=250.1
The Data Comparison that the Collagenase enzyme that the present invention records is lived is in table 2:
| ? | Insoluble collagen albumen | Gelatin |
| Embodiment 1 | 246.2(U) | ? |
| Embodiment 2 | 248.9(U) | ? |
| Embodiment 3 | 251.9(U) | ? |
| Embodiment 4 | ? | 240.3(U) |
| Embodiment 5 | ? | 251.5(U) |
| Embodiment 6 | ? | 250.1(U) |
From table 2 data, Collagenase enzyme activity determination method of the present invention overcomes substrate otherness, and the enzyme recorded precision alive is high, and data consistency is good, and therefore the present invention has significant beneficial effect.
Claims (8)
1. the assay method that Collagenase enzyme is alive, it is characterized in that, described assay method comprises the steps:
1. reagent prepares: prepare the alcoholic solution that concentration is the TrisCl damping fluid of 0.1mol/LpH7.5, the solution of trichloroacetic acid of volume fraction 10%, the o-phthalaldehyde(OPA) solution of 0.6 ~ 0.8mol/L, the hydroxyproline standard liquid of 0.3mmol/L, the acetate buffer of 2mol/L, triketohydrindene hydrate nitrite ion, volume fraction 60%;
2. hydroxyproline sample preparation: pipette respectively hydroxyproline standard liquid in right amount in color comparison tube dilution be settled to same volume, make some hydroxyproline samples that concentration is 0.03 ~ 0.3 μm of ol/mL, to get with the distilled water of hydroxyproline sample same volume in color comparison tube as with reference to sample;
3. hydroxyproline sample determination: add the acetate buffer equal with hydroxyproline sample volume and triketohydrindene hydrate nitrite ion respectively to reference in sample and each hydroxyproline sample, abundant mixing, in 100 ~ 105 DEG C of heating water bath 15 ~ 20min after sealing, cooling, place 10min, then the alcoholic solution dilution of hydroxyproline sample volume 3 times is added, with reference sample for reference solution, the light absorption value of each hydroxyproline sample is surveyed at 520nm place, make C-A typical curve according to the concentration C of hydroxyproline sample and light absorption value A, and simulate typical curve formula;
4. enzymolysis liquid preparation to be measured: take 0.8 ~ 1.2mg collagen substrate, add 0.4 ~ 0.6mlTrisCl damping fluid, in 35 ~ 40 DEG C of preheating 8 ~ 12min, add 0.1ml enzyme liquid to be measured, described enzyme liquid to be measured is long-pending is designated as V
2in 35 ~ 40 DEG C of enzyme digestion reaction Tmin, the value of described T is 25 ~ 35, then add 0.5 ~ 0.7ml solution of trichloroacetic acid to shake up, then the o-phthalaldehyde(OPA) solution adding 0.5 ~ 0.7ml shakes up, and finally adds 0.4 ~ 0.6g silica gel, stirs evenly rear standing 5 ~ 8min, centrifugal supernatant, this supernatant volume is designated as V
1, described supernatant is diluted to concentration 0.03 ~ 0.3 μm of ol/mL as enzymolysis liquid to be measured, extension rate is designated as N;
5. Blank solution: take 0.8 ~ 1.2mg collagen substrate, add 0.4 ~ 0.6ml TrisCl damping fluid, in 35 ~ 40 DEG C of preheating 8 ~ 12min, add 0.5 ~ 0.7ml solution of trichloroacetic acid and shake up, then add 0.1ml enzyme liquid to be measured, in 35 ~ 40 DEG C of enzyme digestion reaction 25 ~ 35min, the o-phthalaldehyde(OPA) solution adding 0.5 ~ 0.7ml again shakes up, and finally adds 0.4 ~ 0.6g silica gel, stirs evenly rear standing 5 ~ 8min, centrifugal supernatant, described supernatant dilution N doubly after as blank solution;
6. the mensuration of enzyme activity: get enzymolysis liquid to be measured and blank solution in right amount in color comparison tube, volume is designated as V
3, add V respectively
3the acetate buffer of volume and triketohydrindene hydrate nitrite ion, fully mix, and in 100 ~ 105 DEG C of heating water bath 15 ~ 20min after sealing, cooling, places 10min, then add 3 times of V
3the alcoholic solution dilution of volume, is reference solution with blank solution, surveys the light absorption value of enzymolysis liquid to be measured at 520nm place;
7., after measuring enzymolysis liquid light absorption value A to be measured, the Hydroxyproline concentration C in corresponding enzymolysis liquid to be measured in the typical curve formula that 3. substitution step obtains, is obtained;
8. the calculating that Collagenase enzyme is alive: computing formula is as follows:
Wherein,
cfor the concentration of hydroxyproline in enzymolysis liquid to be measured;
v 1 for supernatant volume;
mWfor hydroxyproline molecular weight;
nfor supernatant extension rate;
v 2 for enzyme liquid to be measured amasss;
tfor the enzyme digestion reaction time.
2. the assay method that a kind of Collagenase enzyme according to claim 1 is alive, it is characterized in that, step 2. described in refer to 0.1 ~ 1.0ml in right amount, described same volume refers to 1ml, and described concentration is the quantity of the hydroxyproline sample of 0.03 ~ 0.3 μm of ol/mL is 6,7 or 8.
3. the assay method that a kind of Collagenase enzyme according to claim 2 is alive, it is characterized in that, the progressive concentration of described 6 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.18,0.24,0.30 μm of ol/mL; The progressive concentration of described 7 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.15,0.18,0.24,0.30 μm of ol/mL; The progressive concentration of described 8 hydroxyproline samples is followed successively by 0.03,0.06,0.12,0.15,0.18,0.21,0.24,0.30 μm of ol/mL.
4. the assay method that a kind of Collagenase enzyme according to claim 1 is alive, it is characterized in that, described alcoholic solution is ethanolic solution or aqueous isopropanol.
5. the assay method that a kind of Collagenase enzyme according to claim 1 is alive, it is characterized in that, described collagen substrate refers to insoluble collagen albumen or gelatin.
6. the assay method that a kind of Collagenase enzyme according to claim 1 or 2 or 3 or 4 or 5 is lived, it is characterized in that, described enzyme liquid to be measured is the enzyme liquid of the 0.2mg/L be mixed with by finished product Collagenase.
7. the assay method that a kind of Collagenase enzyme according to claim 1 or 2 or 3 or 4 or 5 is lived, it is characterized in that, described silica gel is Kiselgel A.
8. the assay method that a kind of Collagenase enzyme according to claim 1 or 2 or 3 or 4 or 5 is lived, is characterized in that, step 4. middle supernatant is diluted to concentration 0.12 ~ 0.24 μm of ol/mL as enzymolysis liquid to be measured.
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| CN112557522A (en) * | 2019-09-25 | 2021-03-26 | 修正生物医药（杭州）研究院有限公司 | Method for detecting activity of enterokinase |
| CN114231591A (en) * | 2021-12-24 | 2022-03-25 | 河北省微生物研究所有限公司 | Method for measuring activity of collagenase for tanning |
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