In the Laboratory

Chemical Modification of Papain and Subtilisin:

An Active Site Comparison
An Undergraduate Biochemistry Experiment
Mireille St-Vincent and Michael Dickman*
Collge Universitaire de Saint-Boniface, Winnipeg, MB, Canada, R2H 0H7; *mdickman@ustboniface.mb.ca

Enzyme chemistry is an integral part of any introductory biochemistry course and is an important aspect of most
undergraduate protein chemistry courses. By studying the
action of enzymes, students learn to appreciate the dynamic
nature of proteins as well as their role catalyzing myriad
chemical reactions in biological systems. Much time in class,
especially at an introductory level, is spent investigating the
catalytic cycle and the role of the various amino acid residues in the active site of the enzyme. While there are numerous enzyme systems that can be studied, proteases are
often used in class as well as in teaching laboratories because
they are relatively simple, inexpensive, and well understood.
In spite of this, very few undergraduate experiments exist that
directly explore the chemistry of the amino acid residues in
an enzymes active site. (These readers found none.)
Many biochemical techniques such as affinity labeling,
radioactive tagging, and fluorescent and spin labeling take
advantage of the chemical specificity of residue side chains
to probe protein chemistry phenomena (1). Affinity labels
are used to identify active site residues important in catalysis, and spin labeling is employed as a general method for
exploring the conformation of membrane and soluble proteins (2). The reactive side chains of aspartate, glutamate,
lysine, serine, threonine, tyrosine, histidine, and cysteine can
be targeted with a wide variety of reagents.
The experiment we are proposing uses methyl
methanethiosulfonate (MMTS) and phenylmethylsulfonyl
fluoride (PMSF) to specifically modify the cysteine and serine
residues in the active sites of papain and subtilisin, respectively (35). Both enzymes are subjected to the same series
of treatments, and the differences are noted. The chemical
modification of papain by MMTS causes a complete loss of
activity, and when the active site cysteine is regenerated, the
return of activity is noted. Since subtilisin is a serine protease, treatment with MMTS has no effect on it. However,
treatment of subtilisin with PMSF results in an irreversible
loss of activity while the sulfonyl fluoride does not dramatically affect papain under the reaction conditions used in this
experiment.
The comparison of papain and subtilisin accomplishes
many desirable teaching goals. The active site differences between the two proteases are displayed. This opens many possibilities for discussion regarding the reactivity of cysteine
versus serine and the role of cysteine in proteins. (Subtilisin
contains no cysteine while papain has seven.) The covalent
modification of the enzymes and subsequent rescue of papain shows the beginning biochemist that proteins can be
manipulated structurally and that this directly affects their
functioning. In addition, students will appreciate the ease of
manipulating the two enzymes as if they were simple chemi1048

Journal of Chemical Education

cal reagents. As an added plus, substrate specificity can be

discussed, since the enzyme specificities are different, and the
characteristics of the two inhibitors can be compared as well.
All this is possible with a few relatively inexpensive reagents
and a spectrophotometer.
Experimental Procedure
Papain and subtilisin were obtained as crystalline powders from Sigma. MMTS, PMSF, cysteine, and the two substrates, N-benzoyl-DL-arginine p-nitroanilide hydrochloride
and N-succinyl-L-phenylalanine p-nitroanilide, were also
Sigma products.
Stock solutions of the enzymes were prepared from acetate buffer (pH 5.0, 50 mM). Calcium ion was added to
subtilisin by the addition of CaCl2 (10 mM) to the stock
solution as well as immediately after dialysis. Enzyme assays
were conducted in sodium phosphate buffer (pH 7.6, 50
mM) and activity was monitored by measuring the formation of p-nitroaniline at 410 nm. Stock solutions of the substrates were prepared in dimethyl sulfoxide (DMSO), and
the final concentration of DMSO in the assays was 1%. Subtilisin was assayed using N-succinyl-L-phenylalanine p-

cys-25 side chain

COO
S
S

papain

and

NH3

papain

about 90%

SH

about 10%

excess
cysteine

COO
papain

SH

H3N

S
S
COO

almost 100%

NH

cystine

Scheme I. Liberation of papains active site with excess cysteine.

Vol. 81 No. 7 July 2004

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In the Laboratory

nitroanilide while papain was assayed using N-benzoyl-DLarginine p-nitroanilide hydrochloride. MMTS was diluted in
acetonitrile (CH3CN), and PMSF was used as a stock solution in ethanol. All modification reactions were conducted
in acetate buffer at pH 5.0. Dialysis was done in 5 mM acetate buffer at pH 5.0 with 50 mM NaCl using a molecular
weight cutoff of 6,000 to 8,000.
All enzyme solutions and modification reactions were
prepared and conducted in polypropylene tubes fitted with
snap-on polyethylene caps. All enzyme assays were done in
disposable polystyrene cuvettes. The substrate stock solutions
as well as the dilution of MMTS and PMSF were made in
siliconized micro centrifuge tubes. A detailed experimental
procedure is available in the Supplemental Material.W
Hazards
Protein powders are allergenic so students were given
polypropylene tubes with 20 mg of enzyme. They could then
add the acetate buffer directly to the tube at the beginning
of the experiment. CH3CN can irritate eyes, nose, throat,
and skin. DMSO is absorbed directly through the skin and
acts as a solvent carrier for the transport of any substance in
solution. MMTS has a strong odor and is irritating to the
eyes, skin, and respiratory system. In addition, PMSF burns
the skin and eyes, and is very destructive to mucous membranes. Gloves, labcoat, and eye protection should be worn
when manipulating these four chemicals. The solution of
MMTS in CH3CN should be manipulated in a fume hood.
Results and Discussion
Commercial papain often contains a significant quantity of the mixed disulfide of papain and cysteine (6). As a

result, the initial activity of papain can be as low as 10% of

maximum activity (7). For this reason, the enzyme solutions
are first treated with excess cysteine to fully liberate papains
active site cysteine-25. Though this step is not necessary for
the success of the experiment, the activity of papain will seem
to increase 10-fold after chemical modification and subsequent regeneration of the active site cysteine, if it is omitted.
This may confuse students, so it was deemed prudent to first
obtain 100% active papain before modifying with MMTS
(Scheme I). Also, the change from 100% activity to zero activity and back to 100% activity gives a much more dramatic
effect than 10% to zero to 100% activity. If this initial treatment with cysteine is done by the instructor, then the experiment can be easily performed in a three-hour period.
After liberating cysteine-25, the enzymes are assayed with
their respective substrates. Immediately following this step,
both enzyme solutions are split in two samples. MMTS and
PMSF are added to a sample of each enzyme solution
(Scheme II). While a pH of 5.0 appears low for the reaction
between papain and MMTS, it has been shown that cysteine25 of papain and MMTS react at a relatively constant rate
between pH 4.5 and 7.5 (8). PMSF can be used at low pH
to titrate the active sites of serine proteases (9). They are given
half an hour to react before freezing them in either liquid
nitrogen or a dry ice and ethanol bath. (It is important to
quick-freeze protein solutions to avoid enzyme denaturation.)
The frozen enzymes are kept at 20 C until the second period.
After thawing the enzymes at the beginning of the second period, dialysis is done to eliminate the excess MMTS,
PMSF breakdown products as well as any other small molecular weight compounds. Care must be taken during dialysis and opening of the dialysis tubes to avoid introducing
extraneous water into the enzyme solutions, which would

cys-25 side chain

papain

ser-221 side chain

SH

MMTS

MMTS

PMSF

O
S
papain

CH3

O
subtilisin

O
100 % yield

unchanged

and

O
S

OH

subtilisin

100 %

PMSF

O
S

papain

OH

subtilisin

unmodified papain

O
MMTS H3C

CH3

PMSF

CH2

Scheme II. Treatment of papain and subtilisin with MMTS and PMSF.

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Vol. 81 No. 7 July 2004

Journal of Chemical Education

1049

In the Laboratory
Table 1. Student Activity Resultsa for the Reaction between the Substrate and the Enzyme as Measured by the
Absorbance of p-Nitroaniline
Subtilisin and
N-Succinyl-L-Phe p-nitroanilide
Abs

Papain and
N-Benzoyl-DL-Arg p-nitroanilide
hydrochloride
Abs

Commercial enzyme

0.382

0.294

After the first treatment with Cys

0.354

2.134

Enzyme Condition

MMTS

PMSFb

MMTS

PMSFb

After modification with MMTS or PMSF

0.312

0.007

0.016

1.35

After the second treatment with Cys

0.294

0.006

2.09

2.77

The activity is measured by the magnitude of the p-nitroaniline absorbance at 410 nm. Data are the average from five students results.

The comparison with PMSF has not yet been attempted with students. The authors obtained these results.

lower their apparent activity. With the modified enzymes in

hand, another round of assays is performed. This test should
show zero or close to zero activity for papain with MMTS
and for subtilisin with PMSF. No significant change in activity occurs for subtilisin with MMTS, and under the reaction conditions used in this experiment, papain is not
efficiently inhibited by PMSF.
At this point, the modified enzymes are treated with an
excess of cysteine thus reducing the mixed disulfide,
papainSSCH3 as well as any PMSF inhibition of papain.
Neither subtilisin sample is affected by this treatment. A final
assay is performed and the results are presented in Table 1.
Results do vary for two reasons. It is very difficult to
avoid adding water to the enzyme solutions when the dialysis tubes are opened. The additional water dilutes the enzymes, and causes a drop in their apparent activity. This effect
is most easily seen for subtilisin. Also, only one assay is done
at each step owing to the expense of the enzymes and substrates.
Conclusion
The experiment demonstrates how enzymes can be easily modified in a very specific manner. Commercial papain
is brought to near 100% activity, then covalently modified
with MMTS to render it completely inactive and finally reactivated. PMSF has limited effect on papain under the reaction conditions used in this experiment. Subtilisin is
subjected to an identical series of treatments, but the MMTS

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Journal of Chemical Education

has no effect while PMSF completely inhibits this enzyme

irreversibly. The results serve to underline the different active site structures and mechanisms of the two enzymes. This
is accomplished using inexpensive reagents and simple laboratory techniques.
W

Supplemental Material

Detailed instructions for students and notes for instructors are available in this issue of JCE Online.
Literature Cited
1. Fersht, A. Structure and Mechanism in Protein Science; Freeman: New York, 1999; Chapter 9.
2. Columbus, L.; Hubbell, W. L. TiBS, 2002, 27, 288295.
3. Singh, R.; Blttler, W. A.; Collinson, A. R. Methods Enzymol.
1995, 251, 229236.
4. Gold, A. M.; Fahrney, D. Biochemistry 1964, 3, 783791.
5. Whitaker, J. R.; Perez-Villasenor, J. Arch. Biochem. Biophys.
1968, 124, 7078.
6. Klein, I. B.; Kirsch, J. F. Biochem. Biophys. Res. Commun. 1969,
34, 575581.
7. Singh, R.; Blttler, W. A.; Collinson, A. R. Anal. Biochem.
1993, 213, 4956.
8. Roberts, D. D.; Lewis, S. D.; Ballou, D. P.; Olson, S. T.; Shafer,
J. A. Biochemistry 1986, 25, 55955601.
9. Hsia, C. Y.; Ganshaw, G.; Paech, C.; Murray, C. J. Anal.
Biochem. 1996, 242, 221227.

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