EZA—Development of an Enzyme Assay

Summary

This experiment illustrates some of the practical considerations that contribute to the development of a fixed-time (single point) enzyme assay. One key consideration is to maximize the sensitivity of the assay by identifying reaction conditions that use the full working range of the colorimeter (a specialized type of spectrophotometer) during a standard assay. A second consideration will be to determine whether the response of the assay is proportional to the amount of enzyme present in the reaction mixture. In addition, the concepts of units of enzyme activity, volumetric activity, and specific activity will be developed.

Background

Enzyme properties:
Nelson & Cox. "Lehninger Principles of Biochemistry," pp. 190-237
Optional: Ninfa & Ballou. “Fundamental Laboratory Approaches,” pp. 199-218 and
pp. 219-246.

Introduction

An assay is a method to detect the presence of an enzyme. Assays form the basis for many types of enzyme purification schemes. In addition, by varying the assay conditions, investigations of the functional properties of an enzyme, including stability, substrate specificity, and catalytic mechanism can be performed. Depending on experimental considerations, assays can be either fixed-time (single point) or time-resolved. For most enzymes, single point assays can be readily developed, and for some enzymes, this is the only practical method of assay. An essential requirement for a single point assay is that the amount of product formed, or substrate consumed, can be quantitated. Since this type of assay is made after the incubation of enzyme and substrate for a fixed time period, a variety of instrumental approaches may be employed to effect the quantitation. The task of quantitation may be simplified if the enzyme reaction causes a continuous change in the optical absorbance, or in some other readily measurable physical property. Under these circumstances, a time-resolved assay can often be developed.

Whatever method of quantitiation is used for an enzyme assay, the best assays are designed to maximize sensitivity. Achieving high sensitivity may allow the detection of enzyme activity that would otherwise go undetected. For the present experiment, sensitivity is achieved by a combination of choosing the proper substrate and reaction conditions as well as optimization of instrumental conditions. A properly designed enzyme assay can also be used to quantitate the amount of enzyme present in an unknown solution. In order to use an enzyme assay in this manner, it is first necessary to demonstrate that the response of the assay is linearly proportional to enzyme concentration. For the concentration range in which this proportionality holds true, the enzyme assay provides a valid quantitative tool.

The enzyme to be used in this experiment is βgalactosidase from Escherichia coli. This enzyme catalyzes the hydrolysis of a variety of βDgalactosides. The most important natural substrate for this enzyme is lactose, which is cleaved to Dgalactose and Dglucose (FIGURE 1). Lactose is not a conve-nient substrate for the development of an assay, since neither lactose nor the monosaccharide hydrolysis products have an optical absorption in either the visible or the accessible ultraviolet regions. Furthermore, these substances may be rapidly metabolized by other enzymes in biological fluids, leading to underestimation of enzyme activity. In order to allow spectrophotometric measurement to be the instrumental basis for our assay, the chromogenic substrate pnitrophenylβD-galactopyranoside (PNPG) will be used as an alternative substrate.

Upon hydrolysis, PNPG gives Dgalactose and pnitrophenol (FIGURE 2). The pnitrophenol proton has pKa = 7.15, and the ionized phenolate form is yellow-colored (ε400 = 14 mM1 cm1). Since the optimal pH for the βgalactosidase reaction is near neutrality (pH 7.0), only a portion of the pnitrophenol is ionized to the detectable form in the conditions of the enzyme reaction. Complete conversion of pnitrophenol to the detectable phenolate form is accomplished at the end of the reaction time by the addition of sodium carbonate, which raises the pH of the reaction solution to above 10. This pH increase also "quenches" the enzyme reaction by inactivating the enzyme. The colorimetric quantitation of the product can then be made at the analyst's leisure.

Since enzymes are often present in biological solutions in minute quantities, massrelated quantitation is not practical. Instead, enzyme quantities are most commonly described in terms of their catalytic activity: an international unit (I.U., µmol/min) of an enzyme is defined to be the amount of an enzyme that is capable of producing one µmol of product per minute in a standard assay. The enzymatic activity of a particular enzyme solution can be described by volumetric activity, the µmol of product produced per minute by one mL of an enzyme solution; and by a specific activity, the number of units per min produced by an enzyme solution containing one mg of protein

volumetric activity = specific activity =

It should be noted that volumetric activity is dependent on preparation methods and dilution factors, and as such, cannot be used for comparative assessments of enzyme quantities. However, by normalization to the amount of total protein present in a solution, the determination of specific activity allows one to quantitate the amount of enzyme present in a solution in a way that is useful for comparative purposes. For example, during the course of a purification procedure, the specific activity of an enzyme should increase at each step, corresponding to the removal of contaminating, inactive proteins: a homogeneously pure preparation of the enzyme should exhibit the highest specific activity. The conversion between volumetric and specific activities requires that the protein concentration of the enzyme solution be known (mg/mL). In this laboratory exercise, the protein concentration of your enzyme samples will be provided so that you can calculate specific activities. In the GPC laboratory exercise, methods for determining total protein concentration will be introduced.

Materials

— You will be provided a PNPG solution (0.32 mM) and a solution containing βgalactosidase. These solutions are both prepared in 50 mM sodium phosphate buffer, pH 7.0.

Procedure

1. Maximization of the sensitivity of the enzyme assay.
Maximize the sensitivity of the enzyme assay using the following procedure:
(a) Record the protein concentration of the βgalactosidase solution given on the label of the supply bottle in the appropriate place on DATA SHEET I.
(b) Pipet 1.0 mL of 1 M sodium carbonate into each of eight 18 × 150 mm test tubes;
(c) Pipet 12.0 mL of PNPG solution into a suitable reaction vessel (an 18 × 150 mm test tube, for example);
(d) At "zero time", add 4.0 mL of the βgalactosidase solution to the PNPG solution and mix rapidly;
(e) Withdraw 2.0 mL aliquots of the reaction mixture from the reaction vessel and discharge them at carefully timed intervals (1, 2, 3, 4, 6, 8, and 10 minutes) into the tubes containing 1.0 mL of sodium carbonate;
(f) Prepare a blank by adding 1.5 mL of PNPG solution to a tube containing 1.0 mL of sodium carbonate; then add 0.5 mL of enzyme solution. NOTE 1: Be sure to assemble the blank in this manner. Addition of PNPG and enzyme solution without quenching will give a false background. NOTE 2: Buffer is often used in the place of the enzyme solution in assembling the blank, especially if the enzyme solution is available in limited quantities;
(g) Set the colorimeter to operate at 400 nm, adjust to zero absorbance with the blank, and measure the absorbance of your samples in the order that they were prepared. If A400 becomes greater than 1.0, dilute the samples with 3.0 mL of buffer so that A400 < 1.0. If addition of 3.0 mL is not sufficient to provide A400 < 1.0, add another 3.0 mL of buffer. Record the absorbance values and dilutions, if necessary, on DATA SHEET I;
(h) Using the coordinate grid on DATA SHEET I, construct a plot of A400 versus time from your measurements (be sure to account for the dilutions at the longer time periods). Also on DATA SHEET I, plot an "ideal" sensitivity curve, i.e. a straight line from the origin to A = 0.9 at 10 minutes. Determine the slope of this "ideal" curve, and the initial slope of your "real" reaction curve. The ratio of these slopes is your dilution factor. Record both the slopes and the dilution factor on DATA SHEET I.
The significance of the dilution factor: After dilution of your original enzyme solution according to the dilution factor, incubation of 0.5 mL of the diluted enzyme solution with 1.5 mL of the PNPG solution for 10 minutes should give a final A400 value near to, but slightly less than, 1.0. Thus, for a 0.5 mL aliquot of diluted enzyme solution, you will be using close to the full range of the colorimeter. This will have maximized the sensitivity of the assay.
2. Make a single measurement to check your dilution factor.
(i) Using the dilution factor you have calculated, make a single dilution of the original enzyme solution to provide a 0.5 mL aliquot of the diluted enzyme solution. Use this 0.5 mL aliquot and 1.5 mL of PNPG solution to perform an assay in order to ascertain that you have calculated the dilution factor properly. Quench the reaction after 10 minutes and determine the A400 value. You may have to make several tries to get this right.
3. Determine how much of the diluted enzyme solution you will need to complete procedure 4.

Read this introductory part first.

For procedure 4 of this experiment, you will need three different volumes of the diluted enzyme solution to perform validation runs, which will be performed at least in triplicate. An example of a choice of volumes for a validation run would be the following:
1.5 mL PNPG + 0.25 mL diluted enzyme + 0.25 mL buffer = 2.0 mL total.
Obviously, there are many other combinations of volumes of diluted enzyme solution and buffer that will satisfy this relationship. Determine the total amount of diluted enzyme solution needed for your proposed validation experiments. Record the calculations on DATA SHEET II. Have your Teaching Assistant or Instructor review and initial your calculations before making any dilutions of your original enzyme solution.

Now you can dilute your enzyme.

Dilute the proper volume of the original enzyme solution with buffer according to the calculations you have made to make enough solution for the experiments in part 4 and 5. Do not waste the enzyme solution!
4. Using the dilute enzyme solution, perform validation runs according to the procedure outlined below:
(a) Pipet 1.5 mL of PNPG solution into as many 18 × 150 mm test tubes as will be required for your blanks and for the total number of replicates you plan to measure;
(b) Pipet the required amount of buffer into each reaction tube as required and mix;
(c) At precisely timed intervals (1 minute is suggested), pipet the required amount of diluted enzyme solution into the correct reaction tube and mix;
(d) Let the reaction tubes incubate at room temperature. After exactly 10 minutes, stop each reaction by adding 1.0 mL of sodium carbonate to the reaction tube, followed by rapid mixing;
(e) Pipet 1.5 mL of PNPG solution, 0.5 mL of buffer, and 1.0 mL of 1 M sodium carbonate into a 13 × 100 mm test tube to make a blank;
(f) Read the absorbance in each reaction tube in the colorimeter at 400 nm after using the blank to set zero absorbance. Record the absorbance values on DATA SHEET II.
5. Determine the apparent Vmax- and KM-values for your enzyme solution.
You are responsible for the experimental design. Plan to include at least 5 appropriately chosen substrate concentrations (in triplicate).
You will have the following solutions:

Report:

Prepare a cover sheet for you lab report containing your name, section, and title. Include DATA SHEETS I and II, including the original DATA SHEET II signed by a Teaching Assistant or the Instructor. Prepare answers for the following items on additional sheets. Provide sample calculations for questions 2, 4, 7, and 8.
1. For the replicate measurements performed in procedure 4, calculate the mean values, sample standard deviations, and 95% confidence intervals average +/- 2 sigma for your measurements and list them on DATA SHEET II. If you performed your reaction in triplicate or less, the Q-test cannot be used to justify discarding a point. Why is this? If you suspect that any of the individual measurements in a dilution replicate should be discarded, use the Qtest to evaluate this hypothesis. If a data point is discarded, indicate so and the result of Qtest analysis as a footnote to DATA SHEET II.
2. In order determine how many µmol pnitrophenolate is produced by the different volumes of the diluted enzyme solution, assume that the molar absorptivity (ε) of pnitrophenolate is 14 mM1 cm1 at 400 nm, and also assume that the effective path length (b) of the colorimeter cuvettes are 1.0 cm. Use these values to calculate the average µmol of pnitrophenolate produced for each different volume of enzyme solution and enter these values in the appropriate places in DATA SHEET II. Show a representative calculation of how you determined the average µmol of pnitrophenolate.
3. Construct a plot of I.U. (average µmoles of pnitrophenolate formed per minute, not the 10 minute total time of your assay) versus mL of diluted enzyme solution determined in the validation experiment. Examine the plot you have made. In 2-3 sentences describe what range of mL of diluted enzyme solution added to the reaction mixture you consider that your standard assay will give a valid result?
4. You may have noticed that a slight yellow color appears during the enzyme reaction prior to quenching with sodium carbonate. Using the quenched absorbance values obtained in procedure 1 as a starting condition, calculate the absorbance values you would have expected to observed at 1, 2, 3, 4, 6, 8, and 10 minutes if you had run the experiment at pH 7.0 without quenching (assume the pKa of pnitrophenol is 7.15). Add these to the Table in DATA SHEET I in the “predicted” column. Show a representative calculation of how you determined these expected absorbance values.
5. Plot the absorbance and time values calculated for question 4 on DATA SHEET I, and label this plot "calculated, continuous observation."
6. What additional considerations would be important in order to make a continuous, time-based measurement of βgalactosidase activity at pH 7?
7. Calculate the volumetric activity of your diluted enzyme solution (volumetric activity is defined as I.U. per mL of solution). Provide an example of your calculation method.
8. Using the protein concentration of the original β-galactosidase solution, and accounting for the appropriate dilution factors, calculate the specific activity of your diluted enzyme solution and the original enzyme solution in terms of I.U. per mg of protein. Show these values and representative calculations.
9. Provide a table with your measured velocities and substrate concentrations. Make a standard plot of v versus S. From this plot, make a visual estimation of Vmax and KM and report these values on your plot.
10. Make an Eadie-Hofstee plot of the transformed velocity and [S] data ( v = Vmax - ( v / [S])*KM). Use unweighted linear least squares fitting to obtain the best fit line to this plot. Report the slope, intercept and correlation coefficient (r^2) for the fit on the plot. Calculate Vmax and KM and report these values on your plot.
11. Make a one to two sentence evaluation of the results obtained from Question 9 and
Question 10.

Name
Lab section (day)
Group number
Partner’s name

1.  Calculate needed values and fill out Data Sheet II. Answer Q-test part of question.
 

2.  Calculate average micromoles (not micromolar!) of p-nitrophenylate for data from part 4, and fill out Data Sheet II.  Show an example calulation (written by hand is fine).

3.  Plot I.U. vs mL of diluted enzyme soln.  All plots must have title, labeled axes, and units.  All lines must have equation and R squared values, and should go through (0,0) if appropriate.  Please put plots in with your text, not attached at the end.

Comment as your lab book asks.

4.  Show calculation of how you got the predicted absorbances.

Example

Calculation

 5. Graph these predicted absorbances on Data Sheet I and label it.

6.  Answer question, need multiple considerations.
 

7.  Determine volumetric activity of your diluted enzyme solution.  Provide example of your calculation, or just tell me how you got it.
 

8.  Calculate specific activity of your diluted enzyme solution.

Example Calculation

Calculate specific activity of your diluted enzyme solution.

Example Calculation

9.  Table of velocities and substrate concentrations.

Plot of v vs S

Make a visual estimation of Vmax and KM, and report these values.  Use your textbook if you don’t know how to do it.

10.  Eadie-Hofstee plot.  Have plot in text, answer the question.

11.  Evaluate your results for questions 9 and 10.  Do you think you have good data, did something go wrong?  Do the answers agree with each other?  Which plot is more accurate?  Any other thoughts.