In the Laboratory
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Kinetics of Papain
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; *kcornely@providence.edu
Enzyme kinetics experiments are popular in the under-
graduate 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 chymo-
trypsin, 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 Journal of Chemical Education • Vol. 76 No. 5 May 1999 • JChemEd.chem.wisc.edu
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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 experi-
ments 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):

NO2
H O H O
C N C NO2
NH Papain C N C
OH
O
(CH2)3 O +
(CH2)3
NH H2O NH
NH2
C
NH NH2
C
NH
N-α-benzoyl-arginine-p-nitroanilide
(BAPNA)
Upon hydrolysis by a protease, a product is released, p-nitro-
aniline, 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 mea-
suring the rate of formation of p-nitroaniline as a function
of the increase in absorbance of the solution at the λmax of p-
nitroaniline (400 nm) over time at various substrate concen-
trations. 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 avail-
able 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 spectro-
photometer.
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, p-
nitroaniline, may be determined under the conditions
of this assay.
JChemEd.chem.wisc.edu • Vol. 76 No. 5 May 1999 • Journal of Chemical Education
In the Laboratory
Data Analysis
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
NH2 absorbance vs time and obtaining the velocity from the slope
p-nitroaniline of the line in units of absorbance units per minute. If the
(yellow)
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 cata-
lytic 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 under-
standing 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
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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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