A Rapid and Sensitive Spectrophotometric Method

for the Assay of Chymotrypsin*

CHARLES J. MARTIN, JULIUS GOLUBOW, AND A, E. AXELROD

WITH THE TECHNICAL ASSISTANCE OF ALBERT R. FRAZIER

From the Biochemistry Department, University of Pittsburgh, School of kfedicine, Pittsburgh, Pennsylvania

(Received for publication, May 9, 1958)

For several years this laboratory has been concerned with an
investigation of those enzymes in rat skin extracts which, in
part at least, comprise the proteolytic enzyme system of this
organ (l-5). In certain cases, methods have been proposed for
the detection of their individual activities. However, certain
as the test substrate will be presented. Although greater
emphasis will be placed upon the determination of initial re-
action velocities by measuring the rate of p-nitrophenol libera-
tion from this test substrate, it will also be shown that determina-
tions of the rate of substrate disappearance, as measured at

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contemplated studies, e.g. the possible physiological significance
of these proteolytic enzymes in hypersensitivity and post-
thermal injury reactions, have indicated the need for assay pro-
cedures considerably more sensitive than hitherto used (2).
Since the substrate specificities of several of the skin enzymes
resemble that of chymotrypsin in that aromatic amino acid
esters and their acylated derivatives, e.g. n-tyrosine ethyl ester
and N-acetyl-n-tyrosine ethyl ester, serve as susceptible sub-
strates, we attempted to devise a sensitive and rapid spectro-
photometric assay procedure for these enzymes by using chymo-
trypsin as the test enzyme.
Hartley and Kilby (6) first reported that chymotrypsin
catalyzed the liberation of p-nitrophenol from the nitrophenyl
ester, p-nitrophenyl acetate, despite the absence of previously
established structural requirements for a chymotrypsin substrate
in the acyl contributor to the sensitive bond of this compound
(7). Further work by these investigators (8) and by Gutfreund
and Sturtevant (9) led to the concept that a rapid mole per
mole interaction of p-nitrophenyl acetate and chymotrypsin
occurred with resulting formation of acetyl chymotrypsin
the concomitant release of p-nitrophenol (acylation reaction).
This was followed by the much slower deacylation reaction in
which active enzyme was liberated. The actual isolation of
monoacetyl chymotrypsin was accomplished by incubation of
the enzyme with p-nitrophenyl acetate at a pH low enough to
inhibit deacylation (10, 11). Carbobenzoxyglycine p-nitro-
phenyl ester (12) and the nitrophenyl esters of hippuric acid,
hydrocinnamic acid, and others (13) are also hydrolyzed by
chymotrypsin, the efficiency of catalysis increasing as the struc-
ture of the acid moiety approaches that of a typical chymo-
trypsin substrate (13, 14). These observations logically led to
the conclusion that a p-nitrophenyl ester of an acylated aromatic
amino acid should be an extremely sensitive chymotrypsin
substrate. In this paper, details for a simple, rapid, and sensi-
tive spectrophotometric determination of chymotrypsin activity
with the use of N-carbobenzoxy-n-tyrosine p-nitrophenyl
* This investigation. was supported, in part, by Research
Grants A-727 and E-1765 from the National Institutes of Health,
United States Public Health Service and by the Office of Naval
Research under Contract 1833 (00), NR 101-412.
275 rnp, can be utilized with equal facility.
A preliminary note covering some of this material has been
published (15).
EXPERIMENTAL
The purity of the Lu-chymotrypsin preparation’ employed in
these studies was determined by its activity toward ATEE at
25” and pH 8.0 in the presence of 0.10 M CaC& and 0.004 M
tris(hydroxymethyl)aminomethane buffer. The value obtained
for the specific rate constant, JGS,was 3.0 moles per 1. per minute
per mg. of protein N per ml. In an identical system, the maxi-
mal value of 2.87 f 0.10 has been assigned to ka (16). Trypsin
was absent in the chymotrypsin preparation since, at a con-
centration of 0.118 mg. of protein per ml., hydrolysis of Q-N-
tosyl-L-arginine methyl ester was not detected at pH 8.0 in 30
minutes.
Working solutions of chymotrypsin were prepared in 0.12 M
CaCl* (150 to 200 mpg. of protein per ml.) and showed no
diminution in activity upon storage at 25” for 2 days.
and Chymotrypsin concentration was computed from optical
density measurements at 280 rnp by using the relation, milli-
grams of protein per ml. = optical density X 0.486 (17).
Preparation of CTN-5 gm. of purified n-tyrosine ethyl ester
hydrochloride (18) were treated with 3.47 gm. of carbobenzoxy-
chloride in the manner described by Parks and Plaut (19) for
the preparation of ATEE. Ethanol was added to the syrup ob-
tained after the removal of chloroform, and upon addition of
water the material crystallized to yield 4.25 gm. of carbo-
benzoxy-n-tyrosine ethyl ester; m.p., 88.0-88.5’ (this and other
melting points are uncorrected). The reported m.p. is 78” (20).
Saponification of the ester (3.25 gm.) gave 2.64 gm. of carbo-
benzoxy-L-tyrosine; m.p., 94-95“ (reported m.p. 101” (20)).3
1 Lot No. 577-82, Worthington Biochemical Corporation, Free-
hold, New Jersey. Stated to be a salt-free, alpha activation
product (crystallized three times) of chymotrypsinogen, crystal-
ester lized three times.
2 The abbreviations used are: ATEE, N-acetyl-n-tyrosine ethyl
ester: CTN. N-carbobenzoxv-L-tvrosine n-nitronhenvl ester: NP.
p-nitrophenol; NPA, p-nitrophenyl acetate. A ” ’
3 On several occasions, we have obtained crystalline prepara-
tions melting at approximately 105” after crystallization from an

294
February 1959 C. J. Martin, J. Golubow, and A. E. Axelrod 295

Under anhydrous conditions and with stirring, tributylamine

(1.19 ml.) in 5 ml. of dioxane was added to a solution of carbo- t /\ 1
benzoxy-L-tyrosine (1.58 gm.) in 10 ml. of dioxane followed by 0.60

the addition of ethyl chloroformate (0.65 ml.) in 5 ml. of the 1

same solvent. The temperature was maintained at 6-8”. NP
(0.70 gm.) was added 30 minutes later and stirring continued for
1 hour. Solvent was removed in a vacuum. The oil was diluted
with chloroform, washed with cold 1 N' HCI, saturated sodium
bicarbonate, 1 N HCl, and water, and dried over sodium sulfate.
The solvent was then removed in a vacuum. Addition of
methanol to the oil induced crystallization of CTN (yield, 1.0
gm.; m.p., 145”), and after 2 recrystallizations from hot chloro-
form, a white product was obtained that melted at 158-159”.4

Calculated: C 63.34, H 4.62, iS 6.42 260 280 300 320 340 360 380 400 420
Found: C 63.30, H 4.74, N 6.67 Wovelenqth Im,al

[cx]~” + 3.2” (1.0 per cent, in dioxane) and - 16.3” (1.0 per cent, FIG. 1. The absorption spectrum of CTN and its hydrolytic
products. Curve a, CTN at pH 6.5; Curve b, after complete hy-
in acetone). Alkaline hydrolysis released 1 mole of NP per drolysis at pH 6.5; Curve c, after complete hydrolysis at pH 8.0.

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mole of CTN. The CTN concentrat)ion was 3.54 X 10m5M and was added to the
Spectral Absorption Measurements-The absorption spectra of standard reaction mixture (cf. “Experimental”) as a dioxane
CTN at pH 6.5 and of its hydrolytic products at pH 6.5 and 8.0 solution. Hydrolysis of CTN was effected by the presence of 0.20
mg. of chymotrypsin per 3.00 ml. of reaction solution. Blank
are shown in Fig. 1. The absorption maximum for CTN is at
cuvettes contained all components minus CTN.
273 to 275 rnp, a wave length region wherein NPA also has its
maximal absorption (14, 21). The hydrolytic product from
CTN hydrolysis, NP, with a pK, of 7.16 (22), has an absorption 1.00 20
spectrum dependent upon pH. Above pH 7.16 the p-nitro-
phenolate ion dominates with X,,, at 400 rnp (Fig. 1, Curve c) ;
0.80 16
at pH 6.5, NP is mostly in the undissociated form with A,,,, at
x
320 rnp (Fig. 1, Curve b). z
2 120
Under the conditions of the standard assay (see below), the $ 0.60 0
light absorption of ionized NP and of CTN at 400 and 275 rnp, x
x
respectively, was proportional to concentration (Fig. 2, A). .: 0.40 8(O
8
The molar extinction coefficient for CTN ($&, 12,400) was
independent of pH within the range investigated (Fig. 2, B) 0.20 4
whereas e&t”, for NP (as the p-nitrophenolate ion) varied with
pH in agreement with theory (Fig. 2, i?). At pH 8.0, E;\:
equaled 18,750. 2.0 4.0 6.0 6.5 25 8.5
Molar x IO5 PH
Enzyme Activity Measurements-Rate measurements were
FIG. 2. A. Standard curves showing the relationship at pH 8.0
performed in l-cm. quartz cuvettes contained in a thermostated between the optical density and NP or CTN concentration at the
compartment of a Beckman model DU spectrophotometer indicated wave lengths. B. Variation of the molar extinction co-
equipped with a water-jacketed lamp housing. Standard assay efficient of NP and CTN as a function of pH. The S-shaped
mixtures, modified from those reported (15), contained 0.35 ml. smooth curve is the theoretical dissociation curve for NP as cal-
culated from a pK, of 7.15.
of methanol, 1.00 ml. of 0.3 M CaC12, 0.50 ml. of 0.2 tris(hydroxy-
methyl)aminomethane buffer (pH, 8.0), 0.85 ml. of water, 0.20
ml. of enzyme (in 0.12 M CaC&), and 0.10 ml. of substrate solu- blank cuvettes contained water; for measurements at 275 rnp,
tion. The temperature of assay was 30.0’. CTN was usually blank cuvettes contained all components minus substrate. It
dissolved in acetone. However, for absorption measurements was found convenient to prepare a solution sufficient for a
at 275 rnp, dioxane was the solvent because of the large absorp- number of assays which contained all components other than
tion of acetone at this wave length. enzyme and substrate and to add a 2.70 ml. aliquot of this
At an initial CTN concentration of approximately 4 x 10-5 solution to each cuvette. After 5 minutes of equilibration time,
M, at least 5 volumes per cent methanol was necessary for the enzyme was added with introduction of substrate occurring 1
maintenance of solution; at 2.5 X 1O-5 M or lower, methanol can minute later. In general, enzymatic reactions were followed to
be omitted. 30 to 50 per cent of completion.
Reaction solutions for spontaneous rate measurements con- Initial velocities, vo, were calculated from the slopes of optical
tained all components minus enzyme. For readings at 400 mp, density versus time plots and expressed as the change in optical
density per second. The rates of all enzyme-catalyzed reactions
acidified sodium acetate solution. This material did not yield were corrected for the rate of spontaneous hydrolysis determined
the desired condensation product with NP.
under identical conditions. Rate measurements at 400 rnp,
4 The preparation previously used (15) had a melting point of
152” after recrystallization from methanol. In all experiments when performed at pH values other than 8.0, were corrected for
reported here the higher melting product was used. the pH dependence of the molar extinction coefficient of NP.
296 Assay of Chymotrypsin Vol. 234, No. 2

assured by the addition of 0.20 ml. of chymotrypsin (1 mg. per

ml.)) or, when reaction rates were measured at 275 rnp, from the
optical density value obtained by extrapolation of the initial
portion of the rate curve to zero time. The appropriate tmax
value was then used to calculate ao.
a 0.60
RESULTS
x
20 Spontaneous Hydrolysis of CTN-The rate of NP release was
0.40
directly proportional to CTN concentration (Fig. 3, A). The
sensitivity of CTN hydrolysis to pH is shown in Fig. 3, B. It
will be noticed that at pH 6.5 the spontaneous hydrolysis rate
can be considered zero within the time limits requisite for an
enzymatic assay.
Molar x IO5 PH Chymotrypsin-Catalyzed Hydrolysis of CTN-The rate of
FIG. 3. The nonenzymatic rate of CTN hydrolysis as measured CTN hydrolysis was proportional to chymotrypsin concentra-
at 400 mM. A, as a function of concentration at pH 8.0; B, as a tion over the S-fold range explored (Fig. 4, A). The data indi-
function of pH; a~, 3.95 X 10-G M.
cate that an activity determination using approximately 2 mpg.
of chymotrypsin per ml. of assay solution is possible.
Optimal enzymatic activity occurred at pH 8.0 (Fig. 4, B).
This is in agreement with numerous published observations (7,

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23, 24) for, the optimal pH of the a-chymotrypsin-catalyzed
hydrolysis of acylated amino acid esters, amides, hydroxamides,
and hydrazides at approximately 25-30”.
The effect of substrate concentration is given as a plot of
a&a with respect to aa (25) (Fig. 5). K. was estimated to be
3.2 X 1O-5 M from the values of the slope and intercept of the
subjectively drawn line. With a value of 22,000 for the molccu-
lar weight of chymotrypsin (26), the specific rate constant, ka,
was calculated to be 480 sec.-l or, in other units, 8.3 moles of
CTN per 1. per minute per mg. of protein N per ml. The ka
value is higher and the K, value lower than corresponding
constants reported for the chymotryptic hydrolysis of other
acylated amino acid esters (7, 23), although reaction condition
mpg Chymotrypsin per ml PH
differences do not permit direct comparison. Further discussion
FIG. 4. A. The relation between the initial rate of CTN hy-
drolysis at pH 8.0, as measured at 400 rnp, and the chymotrypsin of the effect of the p-nitrophenyl group upon the susceptibility
concentration: an. 3.70 X 10-G M. B. The oH activitv curve for of substrates of the type RCONHCHR1COR2 to chymotryptic
CTN hydrolysis -by chymotrypsin. The o>dinate values are in catalysis will be deferred to a future publication.
arbitrary units and the points are the average of two experiments. Rate Comparison of CTN Disappearance with That of NP
Initial velocities were determined at 400 rns. Formation-For these experiments a dioxane solution of CTN
was used since, at the wave length region of CTN absorption,
even 0.10 ml. of acetone per 3.00 ml. of assay solution produces
intense absorption. However, the use of dioxane in the reaction
solution reduced the rate of chymotrypsin catalysis. For
example, at a chymotrypsin concentration of 13.6 mpg. per ml.
and with a0 equal to 3.80 X 1O-5 M, vo was only 0.7 of the rate
determined in the presence of acetone. The spontaneous
hydrolysis rate was not affected by this solvent difference.
4.0- In Fig. 6, the rate curve for the disappearance of CTN as
measured by the decrease in optical density at 275 rnp is com-
I I I I8 I I I I I pared with the velocity of NP formation. The liberation of
0' I.0 2.0 3.0 4.0 5.0 6.0
NP was followed under identical conditions by the increase in
(lo, Molar x IO5
optical density at 400 rnp. The data indicate that the rate of
FIG. 5. The dependence of the chvmotrvpsin-catalvzed hvdrolv-
sis of CTN upon substrate concentration at pH 8.0. -Data plotted CTN disappearance corresponds to the rate of NP formation.
according to Woolf (25) with initial velocities expressed as the E$ect of Various Proteins, Amino Acid Derivatives, and Re-
change in optical density per second at 400 mH. Chymotrypsin agents upon CTN Hydrolysis-Hartley and Kilby (6, 8) reported
concentration was 6.18 X lo-r0 moles per 1. that although diethyl p-nitrophenyl phosphate and diisopropyl-
phosphorofluoridate completely inhibited the proteolytic, amino
Initial substrate concentrations, a~, were determined either acid e&erase, and amidase activity of chymotrypsin, residual
from optical density measurements at 400 rnp on completely activity toward NPA and p-nitrophenyl ethyl carbonate was
hydrolyzed CTN solutions (complete NP release was rapidly still present. They also reported that NP was liberated from
February 1959 C. J. Martin, J. Golubow, and A. E. Axelrod 297

t&se nitrophenyl esters upon incubation with insulin and prota-

mine and suggested that acylation of free amino or phenolic
bydroxyl groups had occurred. This explanation was supported
by experiments which demonstrated that tyrosine or its ana-
logucs, e.g. ATEE, L-tyrosine ethyl ester, L-phenylalanine ethyl
ester, and others (8) and compounds containing the imidazole
ring (27-30) were capable of liberating NP from NPA. These
observations prompted a study of the effect of various materials
upon the release of NP from CTN.
In these studies rate measurements were determined in a
Bausch and Lomb Spectronic-20 spectrophotometer at 400 rnp
with the use of matched tubes of 11-mm. inside diameter. The
100 0
pH was 8.0 and CTN concentration was at approximately 3.8 X 0 5 IO 15 20 25 30
1O-5 M. Tube contents were equilibrated in a 30°-bath and Minutes
FIG. 6. The rate of CTN disappearance versus NP formation
removed for readings at selected times except in those assays
at pH 8.0. a---@, the decrease in CTN concentration as meas-
involving reaction rates so fast as to necessitate continuous ured at 275 rnp; O--O, the increase in NP concentration as
observation of the increase in optical density. measured at 400 rnp. Dioxane was used as the substrate solvent.
At concentrations of approximately 33 pg. per ml., soybean The enzyme concentration was 13.6 mpg. of chymotrypsin per ml.
inhibitor, lima bean inhibitor, bovine plasma trypsin inhibitor, and a0 was 3.80 X 10m5M.
ovomucoid, lysozyme, salmine, protamine sulfate, heparin,

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pepsin, and chymotrypsinogen (after inhibition of contaminating trypsin for the estimation of activity, and in some cases, ex-
chymotrypsin with diisopropylphosphorofluoridate) did not tensive manipulations or supersensitive light detectors, i.e. a
affect the rate of CTN hydrolysis. Streptokinase (31), at a photomultiplier attachment to a spectrophotometer.
concentration of 1000 units per ml. and fibrinogen, at a con- The use of CTN as a substrate for the assay of chymotrypsin
centration of 200 pg. of clottable protein per ml., were also decreases the required concentration of this enzyme to the
ineffective. millimicrogram range. If one accepts the lower limit of enzyme
L-Tyrosine ethyl ester, L-histidine methyl ester, L-lysine ethyl required as being that amount which will give a total initial
ester, and ATEE, all at 1.3 X 10m3M, produced rate increases of rate equal to twice the spontaneous rate, then at pH 8.0 and
1.4-, 2.3-, 1.2-, and 2.0-fold, respectively. The release of NP with a0 equal to 4 X 10m5M, the activity of 1.5 to 2.0 mpg. of
from CTN by incubation with such compounds probably chymotrypsin per ml. can be readily determined by measurement
occurs by a coupling reaction leading to the formation of carbo- of NP release at 400 mE.c. At a favorable enzyme concentration
benzoxytyrosyl derivatives. Bodansky et al. (32) have de- (approximately 10 mpg. per ml.), optical density versus time
scribed such reactions leading to the synthesis of acylated pep- measurements for the determination of vo need be taken over
tides. only a 60-second time interval or less.
Sulfhydryl compounds induced a rapid liberation of NP from It should be emphasized that the magnitude of the corrections
CT?;. At equimolar concentrations (1.3 X 1O-4 M) cysteine, for the spontaneous hydrolysis of CTN can be minimized or
reduced glutathione, and 2,3-dimcrcaptopropanol produced even eliminated by a decrease in the pH of assay. However,
approximately an 8-fold increase in the rate of hydrolysis. the decreased spontaneous hydrolysis rate obtained by opera-
NPA is also vulnerable to decomposition bp sulfhydryl reagents tion at pH values lower than 8.0 is at the expense of a reduction
(33). in the catalytic efficiency of chymotrypsin (cf. Fig. 4, B) and a
decrease in t$,\$ for the p-nitrophenolate ion (Fig. 2, B). Partial
DISCUSSION
compensation for this loss in sensitivity can be achieved by
Other workers have devised spectrophotometric procedures measurement of CTN disappearance at 275 rnp, since E&:~ for
for the estimation of chymotrypsin activity by utilizing either this compound is independent of pH in the range of values
conventional or specialized substrates in their assay system. For herein discussed (Fig. 2, B). Alternatively, one could resort to
instance, Schwert and Takenaka (18) have applied the technique the measurement of un-ionized NP at 320 rnp as was done by
of differential spectrophotometry to the determination of Dixon and Neurath (14) in their study of the chymotryptic
ahymotrypsin activity using ATEE as the test substrate. Ravin hydrolysis of NPA at pH 5.5.
et al. (34) have proposed the use of benzoyl-m-phenylalanine A number of protein preparations, at relatively high con-
/3-naphthyl ester, although coupling of the liberated ,&napthhol centrations compared to the quantity of chymotrypsin necessary
with tetrazotized diorthoanisidine before absorption measure- for the demonstration of NP release from CTN, were ineffective
ments precludes continuous observation of the course of the in promoting CTN hydrolysis. Thus, it would appear that this
reaction. Optical measurement of the increase in hydroxy- substrate could be utilized to advantage in those systems wherein
brnzoic acid during the chymotrypsin-catalyzed hydrolysis of chymotrypsin-protein inhibitor interactions are under investiga-
normal fatty acid esters of hydroxybenzoic acids has been tion.
advocated as an assay procedure by Hofstee (35). With the As reported (15), trypsin can also function as an efficient
appropriate substrate, the indicator technique of Gutfreund (36) catalyst of CTN hydrolysis. Crystalline papain will also
and of Rhodes et al. (37) for the determination of trypsin activity hydrolyze this substrate (38). It must be emphasized there-
should be amenable to the assay of chymotrypsin. All of the fore, that CTN cannot be considered as a chymotrypsin-specific
above assay procedures require microgram quantities of chymo- substrate. Such data as pertain to the ability of various es-
298 Assay of Chymotrypsin Vol. 234, No. 2

terases to hydrolyze nitrophenyl esters of acylated and non- Acknozuledgments-We wish to thank Mrs. Eleanore Schwartz
acylated amino acids will be the subject of a forthcoming report. for measurements of optical rotation; Dr. J. M. Ruegsegger of
the American Cyanamid Company, Lederle Laboratories
SUMMARY
Division, Pearl River, New York, for a generous supply of
A sensitive method for the determination of chymotrypsin streptokinase; Dr. H. Tauber, Venereal Disease Experimental
activity with quantities of enzyme as small as 4.5 mpg. has Laboratory, United States Public Health Service, University of
been described. This low enzyme requirement was achieved by
North Carolina, School of Public Health, Chapel Hill, North
the use of N-carbobenzoxy-L-tyrosine p-nitrophenyl ester as the
test substrate in conjunction with the techniques of spectro- Carolina, for a preparation of the lima bean inhibitor; Dr. XI.
photometry to measure initial reaction velocities. The proce- Laskowski, Department of Biochemistry, Marquette University,
dure is not only extremely rapid and simple in its execution but School of Medicine, Milwaukee, Wisconsin, for a preparation of
is also suitable for kinetic studies within considerable variation the bovine plasma trypsin inhibitor; and Philip Gottfried, Merck
of reaction solution parameters. Kinetic constants for the and Company, Rahway, New Jersey, for a sample of diisopropyl-
chymotryptic hydrolysis of this substrate have been determined. phosphorofluoridate.
REFERENCES
1. MARTIN, C. J., AND AXELROD, A. E., J. Biol. Chenz., 224, 309 20. BERGMANN, M., AND ZERVAS, L., Ber. deut. them. Ges., 65,
(1957). 1192 (1932).
2. MARTIN, C. J., AND AXELROD, A. E., Biochim. et Biophys. 21. BERGMANN, F., RIMON, S., AND SEGAL, R., Biochem. J., 68,
Acta, 26, 490 (1957). 493 (1958).
3. MARTIN, C. J., AND AXELROD, A. E., Biochim. et Biophys. 22. BENDER, M. L., AXD TURNQUEST, B. W., J. Am. Chem. Sot.,

Downloaded from http://www.jbc.org/ by guest on January 10, 2017

Acta, 27, 52 (1958). 79, 1656 (1957).
4. MARTIN, C. J., AND AXELROD, A. E., Biochim. et Biophys. 23. GREEN, N. H., AND NEURATH, H., in H. NEURATH AND K. C.
Acta, 27, 532 (1958). BAILEY (Editors), The Proteins, Vol. ZZ, Academic Press,
5. MARTIN, C. J., AND AXELROD, A. E., Biochim. et Biophy. Acta, Inc., New York,.l954, p. 1123.
30, 79 (1958). 24. LUTWACK. R.. MOWER. H. F.. AND NIE~~IANN.I C.. I J. Am. Chem.
6. HARTLEY, B. S., AND KILBY, B. A., Biochem. J., 50, 672 (1952). sot., 79; 2179 (1957)‘. ’
7. NEURATH, H., AND SCHWERT, G. W., Chem. Revs., 46,69 (1950). 25. HALDANE, J. B. S., AND STERN, K., Allgemeine Chemie der
HARTLEY, B. S., AND KILBY, B. A., Biochem. J., 56,288 (1954). Enzyme, Theodore Steinkopf, Dresden and Leipzig, 1932,
8.
GUTFREUND, H., AND STURTEVANT, J. M., Biochem. J., 63,656
p. 119.
9.
26. SCHWERT, G. W., AND KAUFMAN, S., J. Biol. Chem., 190, 807
(1956). (1951).
10. BALLS, A. K., AND ALDRIDGE, F. L., Proc. Natl. Acad. Sci. 27. HARTLEY, B. S., Ann. Repts. on Progr. Chem., Chem. Sot.,
u. s., 41, 190 (1955). London, 51, 303 (1955).
11. BALLS, A. K., AND WOOD, H. N., J. Biol. Chem., 219,245 (1956). 28. BRUICE, T. C., AND SCHMIR, G. L., Arch. Biochem. Biophys., 63,
12. DIXON, G. H., AND NEURATH, H., Federation Proc., 16, 173 484 (1956).
(1957). 29. BRECHER, A. S., AND BALLS, A. K., J. Biol. Chem., 227, 845
13. MCDONALD, C. E., AND BALLS, A. K., J. Biol. Chem., 227, 727 (1957).
(1957). 30. BENDER, M. L., AND TURNQUEST, 13. W., J. Am. Chem. Sot.,
14. DIXON, G. H., AND NEURATH, H., J. Biol. Chem., 225, 1049 79, 1652 (1957).
(1957). 31. CHRISTENSEN, L. R., J. Gen. Physiol., 28, 363 (1945).
15. MARTIN, C. J., GOLUBOW, J., AND AXELROD, A. E., Biochim. 32. BODANSKY, M., SZELKE, M., T~M~RKENY, E., AND WEISZ, E.,
et Biophys. Acta, 27, 430 (1958). Chem. and Znd. London, 1517 (1955).
CUNNINGHAM, L. W., AND BROWN, C. S., J. Biol. Chem., 221, 33. DIRKS, B. M., AND BOYER, P. D., Cereal Chem., 28,483 (1951).
16.
34. RAVIN, H. A., BERNSTEIN, P., AND SELIGMAN, A. M., J. Biol.
287 (1956).
Chem., 208, 1 (1954).
17. DREYER, W. J., WADE, R. D., AND NEURATH, H., Arch. Bio-
35. HOFSTEE, B. H. J., Biochim. et Biophys. Acta, 24, 211 (1957).
them. Biophys., 59, 145 (1955). 36. GUTFREUND, H., Discussions Faradau Sot.. 20. 167 (1955).
18. SCHWERT, G. W., AND TAKENAKA, Y., Biochim. et Biophys. 37. RHODES, M.‘B., HILL, R. M., AND FEENEY, ‘R. E., A&Z. bhem.,
Acta, 16, 570 (1955). 29, 376 (1957).
19. PARKS, R. E., AND PLAUT, G. W. E., J. Biol. Chem., 203, 755 38. GOLUBOW, J., AND MARTIN, C. J., Federation Proc., 17, 231
(1953). (1958).
 A Rapid and Sensitive Spectrophotometric Method for the Assay of
Chymotrypsin
Charles J. Martin, Julius Golubow, A. E. Axelrod and With the technical assistance
of Albert R. Frazier
J. Biol. Chem. 1959, 234:294-298.

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