In the Laboratory
Kinetics of Papain
W
An Introductory Biochemistry Laboratory Experiment Kathleen Cornely,* Eric Crespo, Michael Earley, Rachel Kloter, Aime Levesque, and Mary Pickering Department of Chemistry, Providence College, Providence, RI 02918; *
[email protected] Enzyme kinetics experiments are popular in the undergraduate laboratory. In the last decade or so, a wide variety of enzyme experiments have been designed by instructors of biochemistry (1–7 ). These experiments have pedagogic value because they reinforce the concepts of Michaelis–Menten kinetics covered in the lecture portion of the course and give students the experience of calculating kinetic constants from data they themselves have generated. These experiments are also practical because the experiments can be carried out using a single-beam spectrophotometer and thus they are easy to implement. Highly purified enzyme and substrate reagents are commercially available and inexpensive. Protease enzymes (enzymes capable of hydrolyzing peptide bonds) are of interest to biochemists in part because of their physiological roles in dietary protein digestion and cellular protein processing. In general, the mechanisms of protease enzymes are fairly well understood, particularly the enzymes classified as serine proteases, such as trypsin and chymotrypsin, or the thiol proteases, of which papain is the best known. In this investigation, we have chosen to investigate the properties of papain (EC 3.4.22.2), which is called a thiol protease because of the sulfhydryl group in the active site of the enzyme. Thiol proteases occur widely in nature (8). Papain occurs naturally in the papaya, from which the enzyme’s name is derived. The thiol proteases ficin and actinidin are found in figs and kiwi fruit, respectively. The thiol protease bromelain is 644
found in pineapple. The presence of bromelain explains why you can’t add fresh pineapple to a gelatin dessert—the bromelain hydrolyzes the gelatin protein and thus the gelatin doesn’t appear to “set” properly (9). The enzyme has many commercial uses. Papain is often used in the brewing industry to hydrolyze protein, which causes the beer to be turbid. Papain is also used as a meat tenderizer because of its ability to hydrolyze the peptide bonds of the connective tissue proteins collagen, elastin, and actomyosin, which cause meat to be tough. Papain is also used as a cleaner to remove protein deposits on contact lenses. In the undergraduate biochemistry laboratory experiments referenced above, special substrates that produce a colored product were employed so that the progress of the reaction could be monitored spectrophotometrically. This investigation also makes use of a chromogenic substrate (10). Other investigators have studied the kinetics of papain using the nonspecific substrate Azocoll (11). Here we use a more specific substrate, taking advantage of the fact that papain interacts with a phenylalanine residue two amino acids away from the peptide bond cleaved (12). The substrate is Nα-benzoyl-arginine-p-nitroanilide (or BAPNA, for short). This compound was first synthesized in the early 1960s as a chromogenic substrate for trypsin (10), but was subsequently found to be hydrolyzed by papain as well as by some esterases according to the following reaction (13):
Journal of Chemical Education • Vol. 76 No. 5 May 1999 • JChemEd.chem.wisc.edu
In the Laboratory
H C N O
O C
Data Analysis
NO2 NH
(CH2)3 NH C NH2 NH N-α-benzoyl-arginine-p-nitroanilide (BAPNA)
Papain H2O
H C N O
O C
NO2 OH
(CH2)3 NH C NH2 NH
+ NH2
p-nitroaniline (yellow)
Upon hydrolysis by a protease, a product is released, p-nitroaniline, which is bright yellow. Since the substrate and the other product of the reaction are colorless, we can monitor the progress of the reaction spectrophotometrically by measuring the rate of formation of p-nitroaniline as a function of the increase in absorbance of the solution at the λmax of pnitroaniline (400 nm) over time at various substrate concentrations. These data are used to construct a Lineweaver–Burk plot from which the vmax and KM are obtained. Materials and Methods
Reagents and Equipment The source of the enzyme is dried papaya, which is available from Sigma. Although contact lens tablets may also be used as a source of papain for this investigation (11), the dried papaya is less costly. In addition, as contact lens wearers tend to prefer disposable lenses, enzyme tablets containing papain are less widely available. The BAPNA substrate and DMSO solvent are also available from Sigma. This investigation may be carried out using a single-beam spectrophotometer such as a Bausch and Lomb Spectronic-20. However, if the class is small and the equipment is available, superior results can be achieved with a double-beam spectrophotometer. Experimental Procedure Students first prepare stock solutions of the BAPNA substrate by diluting a stock solution of BAPNA with DMSO. They then carry out five kinetic runs and one blank run by mixing the enzyme solution with substrate and monitoring the increase in absorbance at 400 nm over a four-minute time period. Duplicate runs are carried out. A three-hour laboratory period is sufficient for students working in pairs to carry out the kinetic runs. If the instructor wishes to devote a second laboratory period to this experiment, several additional investigations may be carried out. At Providence College, students working in pairs carry out one of the investigations listed below. They then pool their data and each student writes an original report. 1. The protein concentration of the papain solution may be measured by using the Lowry or biuret protein assay, which the students have learned in a previous experiment (14). 2. The dried papaya preparation’s purity may be assessed by SDS-PAGE (15). 3. Data may be collected for a pH–rate profile by carrying out the reaction as described above, using buffers of various pH values. 4. The value of ε for the product in this reaction, pnitroaniline, may be determined under the conditions of this assay.
The depth of analysis will be determined by how many additional investigations are carried out. Minimally, the students calculate the velocity of each of the five reactions by plotting absorbance vs time and obtaining the velocity from the slope of the line in units of absorbance units per minute. If the value for ε was determined, the velocity may be expressed in units of mol L{1 min{1. With these data, the students are able to construct a Lineweaver–Burk plot and calculate the kinetic constants KM and vmax. If the concentration of the enzyme solution has been determined, the students may determine the value of kcat, keeping in mind that the protein concentration is not necessarily the same as the enzyme concentration, which may be verified by SDS-PAGE. If kinetic runs have been carried out at different pH values, students may construct a pH–rate profile. They will be asked to comment on the pH maximum of papain, given what they have studied in class about the mechanism and the protonated states of the catalytic residues His and Cys. Summary This experiment is an investigation of a commercially important enzyme with which the students may have some familiarity. The experiment complements the topic of Michaelis–Menten kinetics that has been studied in the lecture portion of the course and allows the students to construct Lineweaver–Burk plots using their own experimental data. Collection and manipulation of kinetic data increases understanding of the subject of enzyme kinetics. Students don’t always appreciate the complexity of the analysis process when they are given textbook data to construct Lineweaver–Burk plots in homework assignments. Note W Supplementary materials for this article (a student handout and instructor’s notes) are available on JCE Online at http://JChemEd. chem.wisc.edu/Journal/Issues/1999/May/abs644.html.
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JChemEd.chem.wisc.edu • Vol. 76 No. 5 May 1999 • Journal of Chemical Education
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