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1、.Universal Protease Activity Assay: Casein as a SubstrateWritten Protocol:A. AbstractProteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigmas non-specific protease activity assay may be used as a standardized procedure to determine
2、the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin & Ciocalteus Phenol, or Fol
3、ins reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the prot
4、ease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of p
5、rotease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.B. MaterialsReagents:Protease (P4630)Potassium Phosphate, Dibasic, Trihydrate (P5504) Casein (C7078) Trichloroacetic Acid (T0699) Folin & Ciocalteus Phenol R
6、eagent (F9252) Sodium Carbonate, Anhydrous (S2127) Sodium Acetate, Trihydrate (S8625) Calcium Acetate (C1000) L-Tyrosine, Free Base (T3754) 精品.Equipment:0.45 um polyethersulfone syringe filter and syringe 0.45m Dram vials or polypropylene tubes capable of holding 15 mls of solution Spectrophotometer
7、 Cuvettes Pipettes Stir/Hot plate Stir bar ScalepH Meter Graphing ProgramC. Preparation of ReagentsBefore beginning the assay, we need to make sure that the following reagents are correctly prepared:1. A 50 mM Potassium Phosphate Buffer, pH 7.5. Prepare using 11.4 mg/ml of potassium phosphate dibasi
8、c, trihydrate in purified water and adjusting pH with 1M HCl. This solution is placed at 37C prior to use. 2. A 0.65% weight/volume casein solution, prepared by mixing 6.5 mg/ml of the 50 mM potassium phosphate buffer. The solution temperature is gradually increased with gentle stirring to 80-85 C f
9、or about 10 minutes until a homogenous dispersion is achieved. It is very important not to boil the solution. The pH is then adjusted if necessary with NaOH and HCl. 3. A 110 mM Trichloroacetic acid solution, prepared by diluting a 6.1N stock 1:55 with purified water. Trichloroacetic acid is a stron
10、g acid and should be handled with care. 。4. 0.5 M Folin & Ciolcalteas, or Folins Phenol Reagent, which is the solution that will react with tyrosine to generate a measurable color change that will be directly related to the activity of proteases. Folins Phenol Reagent is an acid and should be handle
11、d with care. 5. A 500 mM Sodium Carbonate solution, prepared using 53 mg/ml of anyhydrous sodium carbonate in purified water. 6. An enzyme diluent solution, which consists of 10 mM Sodium Acetate Buffer with 5mM Calcium, pH 7.5, at 37C. This solution is what we use to dissolve solid protease samples
12、 or dilute enzyme solutions. 精品.7. 1.1 mM L-tyrosine Standard stock solution. Prepared using 0.2 mg/ml L-tyrosine in purified water and heated gently until the tyrosine dissolves. As with the casein, do not boil this solution. Allow the L-tyrosine standard to cool to room temperature. This solution
13、will be diluted further to make our standard curve. If necessary, a solid protease sample of predetermined activity, which is dissolved using enzyme diluent to 0.1-0.2 units/ml. This solution serves as a positive control for the quality control assay and as validation for the calculations we will pe
14、rform to determine enzyme activity.D. Setting up the Protease Assay and Standard CurvesTo begin this assay, find suitable vials that will hold about 15 mls. For each enzyme that you will test, you will need 4 vials. One vial will be used as a blank, and three others will be used to assay activity of
15、 three dilutions of the protease. Three dilutions are useful when checking our final calculations against each other. To each set of four vials add 5mls of our 0.65% casein solution, and let them equilibrate in a water bath at 37C for about 5 minutes. Then, add varying volumes of enzyme solution you
16、 want to test to three of the test sample vials, but not the blank. Mix them by swirling and incubate for 37C for exactly ten minutes. The protease activity and consequential liberation of tyrosine during this incubation time is what will be measured and compared between our test samples.After this
17、10 minute incubation, add the 5 mls of the TCA reagent to each tube to stop the reaction. Then an appropriate volume of enzyme solution is added to each tube, even the blank, so that the final volume of enzyme solution in each tube is 1 ml. This is done to account for the absorbance value of the enz
18、yme itself and ensure that the final volume in each tube is equal. Now incubate the solutions at 37C for 30 minutes.During this 30 minute incubation, you may want to set up your tyrosine standard dilutions, which is done using 6 dram vials (dram vials can be substituted with polypropylene tubes) tha
19、t can easily hold 8 mls. To the six vials the 1.1 mM tyrosine standard stock solutions is added with the following volumes in mls: 0.05, 0.10, 0.20, 0.40, 0.50. Dont add any tyrosine standard to the blank. Lower standards may be needed for impure test samples with that will yield little color change
20、. Once the tyrosine standard solution has been added, add an appropriate volume of purified water to each of the standards to bring the volume to 2 mls.After the 30 minute incubation, filter each of the test solutions and the blank using a 0.45 um polyethersulfone syringe filter. Filtration is requi
21、red to remove any insolubles from the samples. The filtration 2 mls of the test samples and blank filtrate is then added to 4 dram vials that can hold at least 8 mls. You can use the same type of vial in which the standards were prepared. To all of the vials containing the standards and standard bla
22、nk, 5mls of sodium carbonate is added, and for best results, 1 ml of Folins reagent is added immediately afterwards. Sodium carbonate is added to regulate any pH drop created by the addition of the Folins reagent. Sodium carbonate is then added to our test samples and test blank. Youll notice that t
23、hese solutions become cloudy after the addition of sodium carbonate. Then, the Folins reagent is added, which will react primarily with free tyrosine. The dram vials are then mixed by swirling and incubated at 37C for 30 minutes.精品.After this incubation, you should notice that the standards have a g
24、radation of color correlating with the amount of tyrosine added; the highest concentrations of tyrosine appearing darkest. You can also notice appreciable color change in our test samples. 2mls of these solutions are filtered using a 0.45 um polyethersulfone syringe filter into suitable cuvettes. No
25、w we performed the assay, we can proceed to the spectrophotometer to record our absorbance values.E. Measuring Absorbance and Calculating Enzyme ActivityThe absorbance of our samples is measured by a spectrophotometer using a wavelength of 660nm. The light path is set to 1cm. Record the absorbance v
26、alues for the standards, standard blank, the different test samples, and test blank. Once all of the data has been collected, we are ready to create our standard curve. In order to generate the curve, difference in absorbance between the standard and standard blank must be calculated. This is the ab
27、sorbance value attributable to the amount of tyrosine in the standard solutions. After this simple calculation, we create our standard curve using a graphing program by plotting the change in absorbance of our standards on the Y axis, versus the amount in micromoles for each of our 5 standards on th
28、e X axis.Volume of Tyrosine StandarduMoles Tyrosine0.050.0550.100.1110.200.2210.400.4420.500.553Once we have entered in our data points, generate a line of best fit and corresponding slope equation.精品.We then find the change in absorbance in our test samples by calculating the difference between our
29、 test sample absorbance and the absorbance of our test blank. Inserting the absorbance value for one of the test samples into the slope equation and solving will result in the micromoles of tyrosine liberated during this particular proteolytic reaction. To get the activity of enzyme in units per/ml,
30、 perform the following calculation.Units/ml Enzyme =(umole tyrosine equivalents released) x (11)-(1) x (10) x (2)11= Total volume (in milliliters) of assay10= Time of assay (in minutes) as per the Unit definition1= Volume of Enzyme (in milliliters) of enzyme used2= Volume (in milliliters) used in Co
31、lorimetric DeterminationTake the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mls, which in our case is 11mls. Then, divide this value by three other quantities: the time of the assay, which we ran for 10 minu
32、tes, the volume of enzyme used in the assay, which was varied, - lets use 1ml - the volume of milliliters used in colorimetric detection, which may differ based on your cuvette. We used 2 mls.Micromoles of tyrosine divided by time in minutes gives us our measurement of protease activity that we call
33、 units. We can cancel out the units for volume measurement in the numerator and denominator, and are hence left with a measurment of enzyme activity in terms of units/ml. In order to determine the activity in a solid protease sample diluted in enzyme diluent we divide our activity in units/ml by the
34、 concentration of solid used in this assay originally in mg/ml. Leaving us with activity in terms of units/mg.Units/mg solid =Units/ml enzyme-mg solid/ ml enzymeF. Conclusion精品.Weve just shown you how to analyze protease activity using Sigmas non-specific protease activity assay. In addition, this a
35、ssay is useful to ensure that our proteases have precisely determined activity before you receive them for your experiments. As you have seen, when doing this procedure its paramount to remember to heat both the casein and tyrosine solutions slowly and not to boil them as boiling will cause degradat
36、ion of the protein and effect results of the assay. Also, its critical to prepare different blanks for both your standards and for each test sample that you have.G. References1. Anson, M.L., (1938) J. Gen. Physiol. 22, 79-892. Folin, O. and Ciocalteau, V., (1929) J. Biol. Chem. 73, 627 如有侵权请联系告知删除，感谢你们的配合！精品