Universal Protease Activity Assay: Casein as a Substrate

A. Abstract

Proteases break peptide bonds. It is often necessary to measure and/or compare the activity of proteases. This non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases for quality control purposes. 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 Folin’s 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 protease. 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 protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.

B. Materials

Reagents:
Protease (P4630)
Potassium Phosphate, Dibasic, Trihydrate (P5504)
Casein (C7078)
Trichloroacetic Acid (T0699)
Folin & Ciocalteu’s Phenol Reagent (F9252)
Sodium Carbonate, Anhydrous (S2127)
Sodium Acetate, Trihydrate (S8625)
Calcium Acetate (C1000)
L-Tyrosine, Free Base (T3754)

Equipment:
0.45 µm polyethersulfone syringe filter and syringe
Dram vials or polypropylene tubes capable of holding 15 mL of solution
Spectrophotometer
Cuvettes
Pipettes
Stir/Hot plate
Stir bar
Scale
pH Meter
Graphing Program

C. Preparation of Reagents

Before 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 phospate dibasic, trihydrate in purified water and adjusting pH with 1 M HCl. This solution is placed at 37°C before 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 for about 10 minutes until a homogenous dispersion is achieved. Do 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 6.1 N stock 1:55 with purified water. Trichloroacetic acid is a strong acid and should be handled with care.
  4. 0.5 M Folin & Ciolcaltea’s, or Folin’s 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. Folin’s Phenol Reagent is an acid and should be handled 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 37°C. This solution is what we use to dissolve solid protease samples or dilute enzyme solutions.
  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 will be diluted further to make the 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 perform to determine enzyme activity.

D. Setting up the Protease Assay and Standard Curves

To begin this assay, find suitable vials that will hold about 15 mL. For each enzyme that you will test, you will need four vials. One vial will be used as a blank, and three others will be used to assay activity of three dilutions of the protease. Three dilutions are useful when checking the final calculations against each other. To each set of four vials add 5 mL of our 0.65% casein solution, and let them equilibrate in a water bath at 37°C for about 5 minutes. Then, add varying volumes of enzyme solution you want to test to three of the test sample vials, but not the blank. Mix them by swirling and incubate for 37°C for exactly 10 minutes. The protease activity and consequential liberation of tyrosine during this incubation time is what will be measured and compared between the test samples.

After this 10 minute incubation, add the 5 mL 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 enzyme itself and ensure that the final volume in each tube is equal. Now incubate the solutions at 37 °C for 30 minutes.

During this 30 minute incubation, you may want to set up your tyrosine standard dilutions, which is done using six dram vials (dram vials can be substituted with polypropylene tubes) that can easily hold 8 mL. To the six vials the 1.1 mM tyrosine standard stock solutions is added with the following volumes in mL: 0.05, 0.10, 0.20, 0.40, 0.50. Don't add any tyrosine standard to the blank. Lower standards may be needed for impure test samples with that will yield little color change. 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 mL.

After the 30 minute incubation, filter each of the test solutions and the blank using a 0.45 µm polyethersulfone syringe filter. Filtration is required to remove any insolubles from the samples. The filtration 2 mL of the test samples and blank filtrate is then added to 4 dram vials that can hold at least 8 mL. You can use the same type of vial in which the standards were prepared. To all of the vials containing the standards and standard blank, 5 mL of sodium carbonate is added, and for best results, 1 mL of Folin’s reagent is added immediately afterward. Sodium carbonate is added to regulate any pH drop created by the addition of the Folin’s reagent. Sodium carbonate is then added to the test samples and test blank. These solutions will become cloudy after the addition of sodium carbonate. Then, the Folin’s reagent is added, which will react primarily with free tyrosine. The dram vials are then mixed by swirling and incubated at 37 ºC for 30 minutes.

After incubation, the standards will have a gradation of color correlating with the amount of tyrosine added; the highest concentrations of tyrosine appearing darkest. You will also notice appreciable color change in the test samples. Filter 2 mL of these solutions using a 0.45 µm polyethersulfone syringe filter into suitable cuvettes. Proceed to the spectrophotometer to record absorbance values.

E. Measuring Absorbance and Calculating Enzyme Activity

The absorbance is measured by a spectrophotometer using a wavelength of 660 nm. Set the light path to 1 cm. Record the absorbance values for the standards, standard blank, the different test samples, and test blank. Once all of the data have been collected, create the standard curve. To generate the curve, difference in absorbance between the standard and standard blank must be calculated. This is the absorbance value attributable to the amount of tyrosine in the standard solutions. After this simple calculation, create the standard curve using a graphing program by plotting the change in absorbance of the standards on the Y axis, versus the amount in micromoles for each of the five standards on the X axis.
 

Volume of Tyrosine Standard  Tyrosine (µmol)
0.05 0.055
0.10 0.111
0.20 0.221
0.40 0.442
0.50 0.553

Once you have entered the data points, generate a line of best fit and corresponding slope equation.

We then find the change in absorbance in the test samples by calculating the difference between the test sample absorbance and the absorbance of the 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, perform the following calculation.
 

Units/mL Enzyme =    (µmol tyrosine equivalents released) x (11)
(1) x (10) x (2)

11= Total volume (in milliliters) of assay
10= Time of assay (in minutes) as per the Unit definition
1= Volume of Enzyme (in milliliters) of enzyme used
2= Volume (in milliliters) used in Colorimetric Determination

Take the number of micromoles tyrosine equivalents released obtained from the slope equation and multiply it by the total volume of the assay in mL, which in this case is 11 mL. Then, divide this value by three other quantities: the time of the assay, which we ran for 10 minutes, the volume of enzyme used in the assay, which was varied, - let's use 1 mL - the volume of milliliters used in colorimetric detection, which may differ based on your cuvette. We used 2 mL.

Micromoles of tyrosine divided by time in minutes gives us our measurement of protease activity that we call 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. To determine the activity in a solid protease sample diluted in enzyme diluent, divide activity in units/mL by the concentration of solid used in this assay originally in mg/mL, which gives activity in terms of units/mg.
 

Units/mg solid =        Units/mL enzyme   
mg solid/mL enzyme

F. Conclusion

This protocol will enable protease activity measurements. In addition, this assay is useful for ensuring that proteases have precisely determined activity before using them for your experiments. When performing this procedure, it's important to heat both the casein and tyrosine solutions slowly because boiling will cause degradation of the protein and affect assay results. Also, it's critical to prepare different blanks for standards and for each test sample.

G. References

  1. Anson, M.L., (1938) J. Gen. Physiol. 22, 79-89
  2. Folin, O. and Ciocalteau, V., (1929) J. Biol. Chem. 73, 627