Determination of protease activity by ultraviolet spectrophotometry
Ultraviolet spectrophotometry for the detection of protease enzyme activity Metric Instrument Co., Ltd. Keywords: ultraviolet spectrophotometry; protease; analysis instrument; UV-1700 ultraviolet spectrophotometer
1. Principle The protease hydrolyzes the casein substrate under a certain temperature and pH condition, then adds the three-stop enzyme reaction, and removes the unhydrolyzed casein precipitate. The filtrate absorbs ultraviolet light and can be determined by ultraviolet spectrophotometry. The enzyme activity was calculated based on the absorbance. Definition of enzyme unit: For every 1 mL of crude enzyme solution, 1 hr of tyrosine is produced as a unit of enzyme activity in 1 min of hydrolyzed casein at a certain temperature and pH, expressed as (u/mL).
2, reagents and solutions
2.1 Three c (CCL3•COOH)=0.4mol/L Weigh three 65.4g, dissolve in water and dilute to 1000 mL.
2.2 Sodium hydroxide solution c (NaOH) = 0.5mol / L According to GB601.
2.3 hydrochloric acid solution c (HCL) = 1 mol / L and 0.1 mol / L
1 mol/L HCL: Dissolve 90 mL of concentrated hydrochloric acid in deionized water and dilute to 1000 mL. 0.1 mol/L HCL: Dissolve 9 mL of concentrated hydrochloric acid in deionized water and dilute to 1000 mL.
2.4 buffer solution
a. Phosphate buffer (pH=7.5) is suitable for neutral protease. Weigh 6.02g of disodium hydrogen phosphate (Na2HPO4•12H2O) and 0.5g of sodium dihydrogen phosphate (NaH2PO4•12H2O), dissolve in water and dilute to 1000mL.
b. Lactic acid buffer (pH=3.0) is suitable for acid protease methyl solution. Weigh lactic acid (80%-90%) 10.6g, dissolve with water and dilute to 1000mL. Sodium lactate (70%) 16g was weighed, dissolved in water and made up to 1000 mL.
Use the solution to take 8 mL of the solution, add 1 mL of the solution, mix well, and dilute twice to form a 0.05% liter/L lactic acid buffer solution.
c, boric acid buffer solution (pH = 10.5) is suitable for alkaline protease A liquid to weigh sodium borate (borax) 19.08g, dissolved in water and made up to 1000 mL. Weigh 4.0g of sodium hydroxide, dissolve it with water and dilute to 1000 mL.
Mix 500 mL of the solution and 400 mL of the solution, and dilute to 1000 mL with water. All of the above buffer solutions must be calibrated with a pH meter.
2.5 10g / L casein solution Weigh 1.00g of casein, accurate to 0.001g, with a small amount of 0.5mol / L sodium hydroxide solution (if the acid protease is used 2 to 3 drops of concentrated lactic acid), add appropriate amount of various A suitable pH buffer solution is about 80 mL. Stir in a boiling water bath while heating until completely dissolved. After cooling, transfer to a 100 mL volumetric flask and dilute to the mark with a suitable pH buffer solution. This solution is stored in the refrigerator and is valid for three days.
2.6 100ug/mL L-tyrosine standard solution
a, weigh 0.100g of L-tyrosine previously dried to constant weight at 105 ° C, accurate to 0.0002g, dissolved in 60mL of 1mol / L hydrochloric acid, and then made up to 100mL, which is 1mg / mL tyrosine standard Solution.
b. Pipette 10.00 mL of 1 mg/mL tyrosine standard solution and dilute to 100 mL with 0.1 mol/L hydrochloric acid to obtain 100 ug/mL L-tyrosine standard solution. This solution is stored in the refrigerator or used immediately.
3 Instruments and equipment
3.1 Constant temperature water bath 40 ± 0.2 ° C.
3.2 American analysis of UV spectrophotometer UV-1700.
4 Analysis steps
4.1 Find the K value. Prepare different concentrations of L-tyrosine standard solution according to the following table. Then, directly measure the absorbance (A) at 275 nm with an ultraviolet spectrophotometer. The absorbance A is plotted on the ordinate and the concentration c of tyrosine is On the abscissa, draw a standard curve (this line should pass the zero point), calculate the amount of tyrosine (μg) when the absorbance is 1 based on the mapping or regression equation, that is, the absorbance constant K value; Should be in the range of (130 ~ 135)). Concentration of tube tyrosine standard solution (μg/mL)
Take the volume of 100ug/mL tyrosine standard solution
mL
Volume of water
mL
Absorbance value Abs. 0 0 0 10 1 10 1 9 2 20 2 8 3 30 3 7 4 40 4 6 5 50
5
5
4.2 Determine the appropriate amount of diluted enzyme solution 2.00mL, casein 2.00mL, three 4.00mL, other operating conditions and the reaction in the Forlin method determination, static precipitation, until filtration. The filtrate was measured for absorbance (A) using a UV spectrophotometer at a wavelength of 275 nm using a 10 mm cuvette.
a, first put the cool solution in a constant temperature water bath of 40 ± 0.2 ° C, preheat for 5 min;
b. Follow the procedure below:
Test tube A (blank) tube B (enzyme sample, need to make three parallel samples) Add enzyme solution 2.00 mL plus enzyme solution 2.00 mL 40 ± 0.2 ° C, 2 min 40 ± 0.2 ° C, 2 min
Add three 4.00 mL (shake) plus casein 2.00 mL (preheated) (shake) 40 ± 0.2 ° C, 10 min (precise timing) 40 ± 0.2 ° C, 10 min (precise timing) plus casein 2.00 mL (pre- Heat) (shake) add three 4.00mL (shake) continue heating for 10min, filter or centrifuge to continue heating for 10min, filter or centrifuge at 275nm wavelength, measure the absorbance of the supernatant at 275nm wavelength, measure the absorbance of the supernatant
c, 1.398 and 166 protease, except for the reaction and the color development temperature of 30 ± 0.2 ° C, the other operations are the same as 4.2. The standard curve is treated the same.
5 Calculate the hydrolysis of casein per minute per ml of enzyme solution at 40 ° C to produce 1 μg of tyrosine, defined as a protease activity unit.
X=A×K×8/2×1/10×n×E=2/5×A×K×n×E
Where: X - the enzyme activity of the sample, (u / mL);
A——the average absorbance of the sample solution; K——the light absorption constant;
8 - the total volume of the reaction reagent, mL; 2 - aspirate the enzyme solution 2.00mL;
1/10 - reaction time 10 min, in 1 min; n - dilution factor
E - conversion factor of ultraviolet method and forint method (neutral, alkaline protease coefficient is 0.50; acid protease coefficient is 0.77).
The results obtained are expressed to integers. 6 The difference in the allowable difference parallel test results shall not differ by more than 3%.
Key words: ultraviolet spectrophotometry; protease; aesthetic analyzer; UV-1700 ultraviolet spectrophotometer
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