59 (1975) 139-146 Publishing Company,
in The Netherlands
A NEW, HIGHLY CHYMOTRYPSIN
AND SPECIFIC ASSAY
and R.M. FLEMING
Departments of Medicine, Sepulveda Veterans Administration 91343 and University of California, Los Angeles, Calif. 90024
May 29, 1974)
1. A simple, highly sensitive, specific fluorometric method for the determination of chymotrypsin is described. 2. The new substrate utilized in this assay, N-glutaryl-glycyl-glycyl-L-phenylalanine P-naphthylamide (GGPNA), is readily soluble in water, stable and highly specific for chymotrypsin. It is not degraded by a large excess of carboxypeptidase B, elastase, thrombin or plasmin and is virtually resistant to trypsin. 3. GGPNA is extremely sensitive to the action of chymotrypsin and permits detection of enzyme concentrations as low as 1 ng/ml. Linearity between enzyme concentration and fluorescence produced is maintained up to at least 3000 ng/ml. 4. (1~~ -Macroglobulin-bound chymotrypsin hydrolyzes GGPNA at a rate about 213 of that exhibited by the free enzyme. 5. Bile pigments in amounts normally found in duodenal juice or traces of blood do not interfere with the assay. 6. GGPNA which releases /3-naphthylamine upon hydrolysis is suitable also for calorimetric and histological determination of chymotrypsin.
Determinations of chymotrypsin in duodenal juice, feces, and other clinical specimens provide useful information in the diagnosis of pancreatic disease [l-3], but lack of a stable, water soluble, sensitive and specific chymotrypsin substrate has made such tests relatively inaccessible. Numerous derivatives of phenylalanine, tyrosine and tryptophan have been used for the determination of chymotrypsin, but none of them satisfies all of these criteria. Amide substrates, yielding p-nitroaniline  or naphthylamine  , are stable and specific, but poorly soluble in water and lack sensitivity. Ester substrates [6-91
on the other hand, some of which are highly sensitive [S,Q] possess a relatively low degree of specificity. Moreover, they are almost water insoluble and many are chemically unstable. In this report we describe the use of a new phenylalanine derivative, ~-gluta~l-glycyl-gIycyl-L-phenylalan~~~e ~-~~aphthylamide (GGPNA), which appears to be an ideal chymotrypsin substrate. Materials and Methods Chymotrypsin A (EC 126.96.36.199), salt free, bovine, A grade from Calbio~hem, La Jolla, California. Trypsin-TPCK, (EC 188.8.131.52) free of chymotrypsin; elastase (EC 184.108.40.206) chromatographically purified, from Worthington Biochemical Corp., Freehold, New Jersey. Plasmin (EC 220.127.116.11) human, lyophilized, from Kabi, Stockholm. Thrombin (EC 18.104.22.168) bovine, from Parke Davis, Detroit, Michigan. Carboxypeptid~e B (EC 22.214.171.124), isolated and purified in our laboratories (unpublished results). ~-glu~~l-glycyl-glycyl”L-phenyl~anine fl-naphthylamide (GGPNA) was synthesized for us by Bachem, Inc., Marina de1 Rey, California and is now available from this source. 2-Amino-2-methyl-1-propanol (AP) from Matheson Coleman and Bell, Los Angeles, California. Tris base and hemoglobin (bovine) from Sigma Chemical Co., St. Louis, Missouri. Bilirubin from Calbiochen~, La Jolla, California. Assay procedure Except where otherwise stated, the following procedure was used: A solution (0.5 ml) of GGPNA substrate (70.0 mg/lOO ml 0.1 M Tris buffer, pH 8.2 containing 2.5 mmol CaCl, 11) and 10 to 50 ~1 of a chymotrypsin solution (5-500 ng enzyme) in 0.0012 M HCl containing 5 mmol CaCl, /l, were made up to a total volume of 1 ml with the above Tris buffer. The mixture was incubated at 37°C for 15 min and the reaction terminated by adding 0.1 ml 1 M citrate buffer, pH 4.5. Blank mixtures from which enzyme hadbeen omitted were run concurrently. After standing at room temperature for lo-15 min fluorescence was measured in an Aminco-Keirs spectrophoto~uorometer (X, X: e2 -Macroglobulin-bound chymotrypsin was 335; hE m : 415 nm, unco~ected). determined in an identical manner. Chymotrypsin activity is expressed in I.U,; 1 mg of chymotrypsin (55% pure by active site titration) corresponded to 0.19 I.U. in this assay. Determination of the fluorescence yield of /3-naphthylamine in our assay system was carried out with a sample of fl-naphthylamine recrystallized from ethanol (caution: carcinogen!).
of clinical samples In order to minimize quenching of fluorescence by bile pigments such as bilirubin and occasional traces of blood in clinical specimens, particularly in duodenal juice, the above assay procedure was modified slightly by terminating the enzymatic reaction with 3 ml of 0.1 M citrate, pH 5.4 instead of 0.1 ml 1 M citrate, pH 4.5. This reduced the quench-factor four-fold.
Results Influence of pH on chymotrypsin activity Fig. 1 illustrates the relationship between
pH and ~hymotrypsin
Fig. 1. Influence
determined AP buffers
of pH on the activity
with GGPNA as substrate. (0.1 M) at 37°C.
in 0.1 M AP buffer at 37’C.
All assays were performed
Relationship between substrate concentration and activity Fig. 2 illustrates the effect of increasing substrate concentrations on the activity of chymotrypsin. The optimum substrate concentration was found to be 6.5 * 10m4 M GGPNA. No inhibition by excess substrate was observed. Fig. 3 presents Lineweaver-Burk plots obtained with two enzyme concentrations. The K, , determined by least squares analysis of the data, was 3.15 * 10Y4 M.
Fig. 2. Relationship
Fig. 3. Lineweaver-Burk
plots of the activity
Fig. 4. Relationship
Assays by standard
Relationship between enzyme concentration and fluorescence intensity This relationship is illustrated in Fig. 4. The sensitivity of the assay is about 5 ng of chymotrypsin per ml. If the period of incubation is extended from 15 to 60 min detection of chymotrypsin at a concentration of 1 ng/ml is possible, Specificity of assay The specificity was investigated by incubation of 10 i.rg of trypsin, 10 pg of elastase and 38 pg of carboxypeptidase B, respectively, in the assay procedure described above. The amounts of these enzymes were about 100-400 times larger than the quantity of chymotrypsin estimated in an average chymotrypsin assay. Fluorescence produced by trypsin and c~boxypeptid~e B was about 0.12% and 0.0016%, respectively, of that calculated for an equivalent amount of chymotrypsin. In the experiment with elastase no reading was obtained, in fact the enzyme appeared to have a slight quenching effect on the fluorescence cont~buted by the substrate. Pfasmin and thrombin when used in very large excess likewise furnished readings slightly lower than the substrate blank. These results are summarized in Table I. When mixtures of 200 ng of chymotrypsin and 10 Erg of trypsin or 200 ng of chymotrypsin and 10 i.tg of elastase were assayed with GGPNA fluorescence yields corresponded to 99% and 96%, respectively, of that obtained with 200 ng of chymotrypsin alone demonstrating the high specificity of the substrate for chymotrypsin. Effect of CC?+and ionic strength Omission of Ca” from the incubation medium decreased enzyme activity by about 17%, but a four-fold increase (10 mM) over that used in the routine assay (2.5 mM) increased activity by only 5%. Addition of NaCl, 0.5 and 1 mol/l, to our assay system decreased fluorescence yields by about 8 and 19% respectively, whereas a further increase of NaCl to 2 mol/l enhanced fluores-
AS A SUBSTRATE
Chymotrypsin Trypsin Elastase Carboxypeptidase Plasmin Thrombin
1 1 1 1 1000 -
12 7.4 3 36
m1.u.** mu*** Casein U NIH U
250.0 0.25* _ 0.15* 0.04t _ 2.75 1.25
* Extrapolated from readings obtained with 10 pg enzyme. ** Assayed with N-benzoyl-L-alanine methyl ester [ 101. * * * Assayed with N-hippuryl-L-arginine [ 11 I. t Extrapolated from readings obtained with 38 ug enzyme. ?‘t N-Benzoyl-DL-arginine p-nitroanilide (BAPNA) possesses little sensitivity for plasmin and thrombin and 1 BAPNA unit therefore represents a much larger amount of these enzymes than 1 BAPNA unit of trypsin.
cence readings by about 9%. Addition of 1 M citrate to terminate enzymatic reaction in the experiments with the highest NaCl concentrations gave precipitates which had to be centrifuged prior to determination of fluorescence. Reproducibility
Two solutions of chymotrypsin were prepared on different days and dilutions thereof subjected to 10 individual concurrent assays by the procedure described under Methods. The mean fluorescence reading for the first solution was 16.5 + 1.3 and that for the second solution 59.2 f 1.3. The standard deviation in 7%of the mean for the two series was k8.2 and +2.3%, respectively. Stability of the substrate
Solutions of substrate in Tris assay buffer, pH 8.2 were kept at 5°C for 3 weeks. A slight increase in the blank readings was observed which, however, did not impair the usefulness of the substrate. Influence
of bilirubin and hemoglobin
A constant amount of chymotrypsin (300 ng, 57.6 p1.U.) was assayed in a series of incubation mixtures containing 5 1-11 of a solution of bilirubin (20-800 mg/lOO ml 0.01 M Na, CO3 ) or hemoglobin (20-2000 mg/lOO ml 0.9% saline). The assays were terminated as described above for clinical samples. Table II summarizes the influence of bilirubin and hemoglobin on the accuracy of the new assay. Readings were virtually unaffected by bilirubin concentrations up to 50 mg/lOO ml and reduced by 15-20% by concentrations of 100-200 mg/lOO ml. The effect of hemoglobin on the assay was less and a concentration of 1000 mg/lOO ml was needed to reduce chymotrypsin recovery by about 13%. Recovery
of bovine chymotrypsin
to human duodenal
Table III presents the results of the determination of chymotrypsin in human duodenal juice before and after the addition of bovine chymotrypsin.
1.r1 of each
in the experiments.
- _ 0
SplUe Chronic Chronic Cancer
pancreatitis pancreatitis of the pancrc :as
Two to 10 ~1 of duodenal juice was used for the determinations and the amount of bovine enzyme added was of the same order as that found in such specimens. The recovery of total chymotrypsin averaged 96%. Discussion The design of our new substrate was based on the analogous trypsin substrate N-carbobenzoxy-glycyl-glycyl-L-arginine @-naphthylamide which is reported to be stable, water soluble, extremely sensitive and highly specific for
trypsin [ 121. In contrast, N-acyl-L-arginine P-naphthylamides which lack the glycyl-glycyl dipeptide show poor water solubility and very low sensitivity [13,14]. On the basis of these observations we reasoned that interposition of a glycyl-glycyl dipeptide between the glutaryl and phenylalanyl residues of the known, but poor chymotrypsin substrate N-glutaryl-L-phenylalanine /3-naphthylamide  should minimize the deactivating effect of the glutaryl group and at the same time increase water solubility. Our expectations were fully realized. The chemical modification increased sensitivity about 1000-fold and resulted in a product of excellent water solubility. Our assay procedure utilizing the new substrate permits detection of chymotrypsin concentrations the sensitivity of the as low as 1 ng/ml (4 * 10-l 4 mol/ml). This approximates most sensitive chymotrypsin assay reported so far which employs the ester substrate N-carbobenzoxy-L-phenylalanyl /3-naphthyl ester  . In contrast to the latter, our GGPNA substrate is highly specific for chymotrypsin. It is hydrolyzed by chymotrypsin at least 1000 times faster than by trypsin and thus is about 15 times more specific for chymotrypsin than the above ester substrate. Carboxypeptidase B, elastase, thrombin and plasmin in overwhelming excess have no effect on the new substrate. A linear relationship between enzyme concentration and fluorescence is maintained over a wide range, several times that shown in Fig. 4. The reproducibility of the assay is excellent and varies no more than +2 to 8%. Solutions of GGPNA in buffer are stable at 5°C for at least 3 weeks. An increase in ionic strength has been reported to enhance the hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide by chymotrypsin as much as 100% . In analogous experiments using the procedure described under Methods and GGPNA as substrate we failed to note similar increases in activity. The presence of Ca*’ (2.5 mM) a s observed previously , enhanced enzyme activity, but a four-fold concentration of Ca” did not result in a further significant increase in activity. GGPNA is hydrolyzed by (Y~-macroglobulin-bound chymotrypsin at approximately 2/3 of the rate observed in assays of the free enzyme and thus is suitable for the detection of chymotrypsin in serum, ascitic fluid and pleural effusions. Using our new substrate we were able to demonstrate small amounts of chymotrypsin in ascitic fluid from patients with acute pancreatitis whereas prior assays of these specimens with N-acetyl-L-tyrosine ethyl ester had failed to reveal even traces of chymotrypsin [ 161. The most important practical application of our method probably is the determination of chymotrypsin in duodenal juice. To determine the potential interference from constituents normally present in such samples we studied the effect of bilirubin and hemoglobin (traces of blood) on our assay. Table II and III show that concentrations of bilirubin up to 50 mg/lOO ml duodenal juice do not interfere with the assay. Bilirubin concentrations of pure gall bladder bile are 50-1000 mg/lOO ml. Quenching of fluorescence by samples of duodenal juice containing excessive amounts of bile can be avoided by suitable dilution of the specimen. The presence of more than traces of blood in clinical specimens is incompatible with the determination of total chymotrypsin. Inhibition of the enzyme by large amounts of (Y,-trypsin-inhibitor in blood is a far greater source of error than the presence of hemoglobin which does not seriously
affect the chymotrypsin readings if concentrations do not exceed 1000 mg/lOO ml. The findings of this study were confirmed by the application of our method to 12 samples of duodenal juice from normal subjects and patients with g~trointestinal disease. Bovine chymot~psin added to these specimens was recovered almost quantitatively (Table III). GGPNA, like other substrates that release naphthylamine, is suitable also for histochemical use or in a modified calorimetric assay. Naphthylamine formed can be reacted with a diazonium salt  or diazotized and coupled with an aromatic amine as described by Blackwood et al. .
Acknowledgement This work was a V.A. Funded
M.R.I.S. No. 7464-05.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
W.P. Dyck, Am. J. Digest. Dis., 12 (1967) 310 R.W. Ammann, E. Tagwercher, H. Kashiwagi and H. Rosenmund, Am. J. Digest. Dis., 13 (1968) 123 J.S. Smith, I. Ediss, M.A. MulUnger and A. Bogoch, Canad. Med. Assoc. J., 104 (1971) 691 B.F. Erlanger. F. Edel and A.G. Cooper, Arch. Biochem. Biophys., 115 (1966) 206 C.E. Blackwood. B.F. Erlanger and 1. Mandel, Anal. Biochem., 12 (1965) 128 G.W. Schwert and Y. Takenaka. Bioehim. Biophys. Acta, 16 (1955) 570 C.J. Martin, J. Golubow, A.E. Axelrod and A.R. Frazier, J. Biol. Chem., 234 (1959) 294 B.H.J. BieLski and S. Freed, Anal. Biochem., 7 (1964) 192 E. Haas, Y. Elkana and R.G. Kulka, Anal. Biochem., 40 (1971) 218 H. Rinderknecht, P. Silverman and M.C. Geokas, Enzymologia, 40 (1971) 345 J.E. FoIk, K.A. Piez, W.R. Carroll and J. Gladner, J. Biol. Chem., 235 (1960) 2272 G.F. B&&a and W.B. Quay, Enzyme, 12 (1971) 311 A. Riedel and E. Wiinsch. Z. Physiol. Chem., 316 (1959) 61 M.M. Nachlas, R.E. Plapiger and A.M. Seligman, Arch. Biochem. Biophys.. 108 (1964) 266 H. Rinderknecht, P. Silverman. M.C. Geokas and B.J. Haverback, Clin. Chim. Acta, 28 (1970) 239 J. Bietb, P. Metais and J. Warter, Enzyme, 13 (1971) 13 S.P. Kramer, R.E. Plapiger, P.D. Bharadwaj, H.H. Platt and A.M. Seligman, J. Surg. Res., 8 (1968) 253