Title: Enzymatic Activity

Techniques to Master:

  1. Perform enzymatic activity assays
  2. Produce and interpret progress curves
  3. Measure reaction velocity
  4. Calculate specific activity

Learning Objectives:  

  1. Use a spectrometer or plate reader to measure rates  of the enzyme-catalyzed reaction.
  2. Design experiments to optimize substrate concentration, enzyme concentration,  pH and, in some cases, the appropriate buffer and cofactor concentration.
  3. Calculate specific enzyme activity using enzymatic rates,  enzyme concentration, and protein concentration.

Background: 

A. Enzyme activity assays determine enzyme presence and activity level. Enzyme activity assays usually follow the appearance of a product or the disappearance of a substrate over time. An easy way to monitor the reaction is by choosing a substrate that contains a group that, when removed from the substrate by the enzyme, can be monitored spectrophotometrically. One example of such a substrate for a hydrolase is p-nitrophenyl acetate (PNPA), which can be hydrolysed into acetate and p-nitrophenol (PNP) (Figure 1). p-nitrophenyl acetate is colorless and PNP is yellow. We can follow this hydrolysis reaction by monitoring the increase of absorbance at 405 nm using a spectrophotometer (or 96-well spectrophotometric plate reader). Enzyme activity assays are performed by zeroing a spectrophotometer with a cuvette containing a known concentration of substrate and an appropriate buffer. A small amount of enzyme is added to the cuvette and then the reaction is monitored by measuring the change in absorbance over time (Figure 2). This plot is also known as a progress curve. In the case of the p-nitrophenyl acetate substrate, the absorbance would correspond to the release of PNP from the substrate through the action of the enzyme. Progress curves may not be linear near the beginning of the reaction but rather may show a lag in product production (Figure 3) or a burst of activity that later in time becomes linear (Figure 4). Besides absorbance, chemiluminescence, and fluorescence can also be used to monitor reactions. 

B. Beer’s Law. The absorbance values obtained can be converted into the concentration of the product in each cuvette at a given point in time. To do this, it is important to understand Beer’s Law given in Equation 1:

 

The extinction coefficient is a number specific to the product being studied at a specific wavelength. For example [1], PNP measured at 400 nm has an extinction coefficient of 18,320 cm-1 M-1. Each absorbance measurement is converted to the concentration of product in the cuvette and plotted (Figure 5). The slope of the graph shows the change in product concentration over the change in time (Δ[P]/Δt) or the velocity of the enzyme-catalyzed reaction. This visualizes how much of the substrate is being converted to product over time.

For example, if the linear portion of the progress curve has a slope Δ[A]/Δt = 0.100/min and if the cuvette pathlength is 1.0 cm (typical), then dividing Δ[A]/Δt by el gives, 0.100/min/(18,320 cm-1M-1 x 1.0 cm) = 5.5 x 10-6 M/min for the velocity, V.

C. Specific Enzyme Activity. By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme that can transform 1 mmol of substrate per minute at 25 ºC.  Enzyme activity refers to the total amount of units in the solution. Using the above example, the activity based on the molecular weight of PNP = 139.1 g/mol would be 759 micromol/min or 759 units of enzyme activity.

This is a useful term if one is simply determining if active enzyme is present, but is not useful when comparing different solutions of enzyme.  Comparing different solutions of enzyme is important when assessing the progress of an enzyme purification. To do this, one uses specific activity, which is defined as the units of enzyme per milligram of total protein (units/mg). Specific activity informs the scientist to the purity of the enzyme.  The purer the enzyme solution the higher the specific activity. Protein concentrations are measured using techniques such as the Bradford assay.

Note that the milligrams of protein represents both active enzyme, inactive enzyme, and any other proteins in the solution but that the units of activity reflect only the active enzyme.

D. Percent recovery and Fold Purification

In addition to specific activity, enzyme purification at each step of a purification procedure can be quantified by calculating the percent recovery and fold purification.

Percent recovery is f the total units at any step along the purification scheme divided by the total units of activity in the crude, unpurified protein material. Fold purification is the ratio of the specific activity of a fraction to the specific activity of the crude material.

Purpose: The purpose of the enzyme activity assay is to provide evidence for the function of the protein. Measurement of enzyme activity tests the hypothesis of the protein’s function based on computer modeling of the project. If the protein has retained activity and not been denatured during isolation and purification and if the proper substrate has been selected then one can conclude the protein has the predicted function. Questions will remain as to the in vivo function of the enzyme and in what cellular pathway(s) the enzyme functions if it came from a living cell and was not engineered.

Experimental Design Considerations:

In setting up an enzyme activity assay one should bear in mind the following:  How soluble are both the enzyme and the substrate?  How likely is it that the enzyme has been denatured during its purification?  How will the course of the reaction be monitored - through appearance of products or disappearance of substrate or cofactors? What type of spectroscopy will be used (ultraviolet, visible,  fluorescence)? In what order will the reagents be mixed? Substrate added to enzyme or vice versa?  What are the enzyme and substrate concentrations that will produce linear progress curves?  How important are pH, buffer concentration, cofactors, metals, and temperature?  Looking at the source organism for the gene may inform choices of pH, temperature, anaerobic conditions for the assay (where does the organism live?). What blanks and controls need to be prepared?

Supplies:

        Equipment:

        UV Visible Spectrophotometer

        96-well plate reader

        Cuvettes or 96-well plates

        Pipettes and tips

        Containers for dilution

        pH meter

        Reagents:

        Buffer standards (4, 7, 10)

        Ultrapure water

        Ice bath

        Stock solutions of substrates, enzymes, and buffers

Safety Considerations: Consult MSDS and bottle labels for safe handling of proteins, substrates and buffers. Wear appropriate personal protective equipment.

Procedure:

Most students will find it helpful to perform an activity assay on a known enzyme prior to or in concert with their unknown protein. Since many unknowns in this project are serine proteases the following procedure is based on assay of the activity of ɑ-chymotrypsin (I.U.B.: 3.4.21.1). The substrate used in the assay is PNPA. The procedure, taken from the article by Kezdy and Bender [1], produces first-order kinetics in enzyme under the conditions where the initial substrate concentration is much greater than the enzyme concentration. In an abbreviated form the procedure is:

  1. Prepare pH 7.8 buffers: 0.03 M phosphate or 0.05 M Tris-HCl buffer (pKa = 8.3) in 1.6 to 4.0% acetonitrile/water solution. The acetonitrile solution helps to solubilize the substrate. Ghosh [3] ran assays in 10 mM Tris/HCl at pH = 7.5 but at 30oC compared to 25oC in Kezdy.
  2. The buffer solution (3.00 mL) is maintained at 25.0  0.5oC in a 1.0 cm UV-compatible cuvette.
  3. The PNPA ester solution (50 microliter) in buffer is made fresh and is added to the buffer in the spectrometer and the spontaneous hydrolysis measured for about 4 minutes. The initial concentration of the substrate is designed to be much greater than that of the enzyme to insure first-order kinetics when the enzyme is added. The substrate (MW = 181.2) concentration according to Kezdy can be anywhere from about 48 x 10-5 M to 280 x 10-5 M, i.e., 0.087 mg/mL to 0.51 mg/mL. A 5-10 mg/mL stock solution of PNPA can be prepared and diluted appropriately. Keep PNPA solutions in the dark to avoid excessive decomposition prior to the assay. Ghosh [3] observed linear time dependence during the first 5 min at all PNPA concentrations which ranged from 0.04 mM to 0.13 mM (0.0072 mg/mL to 0.023 mg/mL when catalyzed with 11 x 10-6 M enzyme.
  4. Then 50 microliters of enzyme solution in buffer is added to the cuvette containing the substrate to start the hydrolysis reaction. The enzyme concentration is 5.7 x 10-5 M  0.1 x 10-5 M, i.e., 1.4 mg/mL based on a molecular weight for chymotrypsin = 24.8 kDa. Its isoelectric point pI = 8.8.
  5. The absorbance of the solution is measured at 400 nm for several minutes in  order to determine the slope of the linear region of the reaction. If the reaction is too fast leading to saturation of absorbance then reduce the amount of enzyme added. If the reaction is too slow then add more enzyme solution. Ghosh [3] calculated the actual enzymatic hydrolysis by subtracting the non-enzymatic rate from the total reaction rate but noticed no variation in absorbance at 400 nm for PNPA hydrolysis in Tris/HCl buffer within 5 minutes of initiating the reaction.

Clean-up: If using quartz cuvettes they should be carefully cleaned with non abrasive cleaners to avoid scratching the cuvette surface. If a 96-well plate reader is used DO NOT leave used plates in the instrument after collecting data as the optics can be adversely affected. Reactions mixtures and unused substrates should be disposed of properly following MSDS guidelines.

        

Interpreting Results: 

  1. From the progress curve find the linear region and determine the slope,  Δ[A]/Δt where time is in minutes.
  2. Using the known extinction coefficient for the absorbing species (18,300 M-1cm-1 for PNP) convert the slope of the progress curve from Δ[A]/Δt to Δ[P]/Δt using Beer’s Law, A = ecl. Where c is the molar substrate concentration [P]. The cuvette pathlength l is usually 1 cm, therefore, Δ[P]/Δt  = ( Δ[A]/Δt ) / 18,300. Note: Ghosh [3] reported the molar extinction coefficient of the PNP anion as 18,000 M-1cm-1 at 400 nm.
  3. Convert to activity in units of micromol/min by multiplying Δ[P]/Δt by 1 x 106 micromol/mol  x VF where VF is the volume of the assay in liters which in the above procedure is 0.0031 L.
  4. Calculate the relative activity of the enzyme, by dividing the activity by the volume of enzyme used in the assay. In the present example this would be 0.050 mL to give units of activity of micromol/min/mL.
  5. Calculate the specific activity by dividing the relative activity by the enzyme concentration (mg enzyme / mL) used in the assay. In the chymotrypsin example this is 1.4 mg/mL.

Hypothetical Sample Calculation:

  1. Slope of linear portion of progress curve, absorbance per minutes =  Δ[A]/Δt = 0.400 / min
  2. Change in product molarity per minute Δ[P]/Δt  =  V  =  (0.400 /min) / (18,300 M-1cm-1 x 1.0 cm) = 2.19 x 10-5 M/min. Ghosh3 observed a maximum reaction velocity of ca. 0.035 x 10-6 M/min with 11 x 10-6 M enzyme and 0.13 mM PNPA at 30oC. This maximum velocity would correspond to an observed Δ[A]/Δt = 0.001/min. The catalytic rate constant was calculated to be kcat = 4.71 x 10-3 s-1 and the Michaelis-Menten constant was KM = 5.22 x 10-5 M.
  3. Activity = 2.19 x 10-5 M/min x 1 x 106 micromol/mol x 0.0031 L = 0.0678 micromol/min.
  4. Relative activity = 0.0678 micromole/min/0.050 mL = 1.36 micromol/min/mL.
  5. Specific activity = relative activity / enzyme concentration = (1.36 micromol/min/mL) / (1.4 mg/mL) = 0.97 micromol/min/mg.
  6. If preferred, the specific activity can be expressed in nanomol/min/mg by multiplying by 1000, i.e., specific activity = 970 nanomol/min/mg.

References:  

  1. Kezdy, Ferenc J. and Bender, Myron L. (1962), Biochemistry, Vol. 1, No. 6, 1100.
  2. Farrell, S. O., and Taylor, L. E. (2006) Experiments in Biochemistry: A Hands-On Approach: A Manual for the Undergraduate Laboratory, 94-95.
  3. Ghosh, Kallol K., and Verma, Santosh, K. (2008), Indian Journal of Biochemistry and Biophysics, Vol. 45, 350-353.
  4. Ninfa, A. J., Ballou, D. P., and Benore, M. (2009) Fundamental Laboratory Approaches for Biochemistry and Biotechnology, 2nd Ed., Wiley, Hoboken, NJ
  5. Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis, 2nd Ed., Verlag Chemie; Academic Press, Weinheim; New York

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