Serum carboxypeptidase B: A spectrophotometric assay using protamine as substrate

ANALYTICAL

BIOCHEMISTRY

73, 41-5 1 (1976)

Serum Carboxypeptidase 6: A Spectrophotometric Assay Using Protamine as Substrate1p2 NEAL

C. CORBIN~, TONY E. HUGLI~,

Department

of Molecular

Immunology, La Jolla,

AND HANS J. MULLER-EBERHARD~

Scripps California

Clinic 92037

and Research

Foundation,

Received November 3. 1975; accepted December 16, 1975 A quantitative calorimetric assay for serum carboxypeptidase B (SCPB. anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminal lysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and CSa, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl, before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippurylarginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the calorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 3 18 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.

Serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is an cY-globulin and possesses a functional specificity similar to that of bovine pancreatic carboxypeptidase B. The enzyme has been purified from serum and its physical properties have been previously described by Bokisch and Mtiller-Eberhard (1) and by Oshima and Erdos (2). SCPB inactivates the complement derived anaphylatoxins C3a, and C5a, as well i This is publication number 1012 of Scripps Clinic and Research Foundation. * This work was supported by United States Public Health Service Grants Al 07007 and HL 1641 I and American Heart Association, Inc.. Grant-In-Aid 74-864. 3 Dr. Corbin is supported by United States Public Health Service Training Grant 5 Tl GM 683. * Dr. Hugli is supported by American Heart Association, Inc., Established Investigatorship 72-175. 5 Dr. Miiller-Eberhard is the Cecil H. and Ida M. Green lnvestigator in Medical Research, Scripps Clinic and Research Foundation. 41 Copyright All rights

0 1976 hy Academic Pre\\. Inc. of reproductmn in any form revswed.

42

CORBIN,

HUGLI

AND

MOLLER-EBERHARD

as bradykinin and kallidin, by removing the basic COOH-terminal amino acid residue from these peptides. SCPB presumably serves as the major regulatory enzyme of circulating vasoactive peptides. Since deficiencies in SCPB may be associated with disease, it will be important for the clinical investigator to have available a procedure for the quantitation of the enzyme. A specific and quantitative assay was therefore developed for the determination of carboxypeptidase activity in the serum from diseased and healthy individuals. The assay utilizes an inexpensive and commercially available protamine (salmine) as a substrate rather than the synthetic carboxypeptidase B substrates, hippuryllysine and hippurylarginine, which were introduced by Folk er al. (3). Incubation of salmine with serum released only arginine, indicating that the digestion was highly selective and involved only SCPB. The release of arginine was quantitated calorimetrically with a ninhydrin reagent developed by Yemm and Cocking (4) and modified by Matheson and Tattrie (5) to differentiate between the amino groups of proteins and amino acids. A quantitative comparison was undertaken of the SCPB activity in normal human sera and sera from patients in clinical shock associated with activation of the complement system (6,7). MATERIALS

AND METHODS

Substrate

Salmine was purchased from Calbiochem, San Diego, California. Ninhydrin was obtained from Pierce Chemical Co., Rockford, Illinois. All other chemicals were obtained from standard commercial sources and were of the highest purity available. Human

Sera

The sera from patients with shock after dextran (Macrodex)6 infusion were kindly supplied by Dr. Karl Arfors, Uppsala, Sweden. The sera from patients with the shock syndrome associated with Dengue fever’ were obtained through a World Health Organization study conducted by investigators of Scripps Clinic and Research Foundation, La Jolla, California (8,9). Sera from cystic fibrosis patients were supplied by Dr. Theodore Friedman, University of California, San Diego, and Dr. Alexander G. Bearn, Cornell University Medical College, New York. 6 Macrodex: Trade name for dextran commercially prepared by Pharmacia AB, Uppsala, Sweden. ’ Dengue shock syndrome or hemorrhagic shock syndrome is a complication of Dengue hemorrhagic fever. Dengue hemorrhagic fever results from an arbor B type viral infection and is endemic in Southeast Asia. Complement has been implicated as a mediator in the development of this clinical shock syndrome.

SERUM

CARBOXYPEPTIDASE

B (SCPB) ASSAY

43

Amino Acid Analysis

Amino acid analyses were performed using a Beckman amino acid analyzer (Spinco Model 121) according to the method described by Spackman, Stein, and Moore (10) and modified for accelerated analyses by Spackman (11). Protein samples were placed in Pyrex tubes containing 1 ml of constant boiling HCl and the tubes were evacuated and sealed. Hydrolysis was performed at 110°C for 24 hr. Preparation of the Ninhydrin Reagent

The ninhydrin reagent was prepared by adding 30 ml of 5% (w/v) ninhydrin in methyl cellusolve to 150 ml of a 1 x lop4 M solution of NaCN in methyl cellusolve. This reagent was stored at 4°C for at least 12 hr before being used. It was shown that the reagent was stable for at least 10 days when kept in a brown bottle at 4°C under nitrogen. Assay procedure

The assay was performed using serum diluted 1:4 with saline (0.85%, w/v) and made 5 mM in CoCl,. After standing at 24°C for 2 hr, 50 ~1 of serum was combined with 150 ~1 of saline (O.SS%, w/v) and 100 ~1 of salmine solution was added (10 mg/ml in 0.03 M HEPES8, pH 7.2). This mixture was incubated for 30 min at 37°C and digestion was terminated by adding 25 ~1 of aqueous TCA (12%, w/v). The mixture was allowed to stand at least 10 min and then heated at 95 to 100°C for 5 min. The mixture was then centrifuged at 4,000g for 3 min in a Beckman Microfuge B and 200 ~1 of the supernatant was removed for analysis. The supernatant was combined with 0.8 ml water, 0.5 ml citrate buffer (0.2 M, pH S.O), and 1.2 ml of the ninhydrin reagent. The total assay mixture was heated for 7.5 min at 95 to 100°C and then immediately cooled in ice for 5 min according to the method of Matheson and Tattrie (5). The samples were centrifuged 400g for 7 min and 1.0 ml was removed and diluted to 2.0 ml with 1 M NaCl. The absorbance was measured at 570 nm. Definition of a Unit of SCPB Activity

A unit of SCPB activity is defined as 1 nmol of arginine released per minute per milliliter of serum at 37°C as determined by direct amino acid analysis of the digestion mixture. Protein determination

The method of Lowry et al. (12) was used. K HEPES, N-Z-hydroxyethyl-peperazine-N’-2-ethanesulfonic line)-ethanesulfonic acid.

acid; MES, 2-(N-morpho-

44

CORBIN.

HUGLI

AND MULLER-EBERHARD TABLE

1

AMINO ACID COMPOSITION OF SALMINE~ Residues/mole Amino acid Arginine Serine’ Proline Glycine Alanine Valine Leucine

Nanomols 150.0 22.4 17.2 15.8 4.9 12.6 2.6

Found

Expected*

22.7 3.4 2.6 2.4 0.7 1.9 0.4

21-22 4 2-3 2 o-1 2 0

n Commercial protamine preparations obtained from salmon sperm contain several nearly identical polypeptides. Each component contains an NH,-terminal proline and four arginines at the carboxy terminus. b Based on an assumed molecular weight of 4300 (13). c Corrected for loss during hydrolysis for 24 hr at 110°C in ~vmw.

RESULTS

Stability

of Salmine

as a Substrate

In order to show that salmine was a suitable substrate, it was examined in the following manner. An amino acid analysis showed that the composition of the salmine use for these assays (Table 1) agreed with that of 225

180 %35 t= 90 45

Time [minutes) FIG. 1. Digestion of salmine by SCPB results in a selective release of arginine. Values of free amino acids are given for arginine, glutamic acid, glycine, and valine. Salmine (240 nmol) was incubated with 12.5 ~1 of cobalt treated serum in a total volume of 300 ~1. The incubation was carried out in 0.03 M HEPES at 37°C and pH 7.2. The quantity of arginine released after 30 min corresponds to 0.56 mol of arginine per mol of salmine present.

SERUM

CARBOXYPEPTIDASE

I 200

B (SCPB) ASSAY

, 400 600 ~6 Protrmins

600

, 1000

45

, 1200

FIG. 2. Extent of arginine release at various salmine concentrations. The reaction mixture containing 12.5 ~1 of serum was incubated for 30 min at 37°C and pH 7.2. The quantity of substrate necessary to saturate the enzyme is approximately 600 pg. The quantity of substrate present under standard assay conditions is I mg (8 x IO-’ M).

previously published analyses (13). No free amino acids were detected in the unhydrolyzed material. Amino terminal analysis was performed by direct Edman degradation (14) and only proline was detected by amino acid analysis following hydrolysis in hydroiodic acid. A known quantity of salmine was digested with bovine pancreatic carboxypeptidase B and the amino acid analysis showed that only arginine was released. The total quantity of arginine release corresponded to four TABLE INFLUENCE

OF

CoCI,

ON

2

SCPB

ACTIVITY

IN SERUMS

Incubation in 5 mM CoCI, 0-d 0.0 0.5 1

2 4 6 336

0.04 0.17 0.19 0.24’ 0.28 0.31 0.31

a Human serum (12.5 ~1) was incubated in the presence of 5 mM CoCl, at 25°C for various intervals. The total SCPB activity was then estimated by the assay procedure described in the text. b Change in ninhydrin color development. c Two hours of preincubation with CoCI, was selected as a standard assay condition for the enhancement of SCPB activity in serum.

46

CORBIN,

HUGLI

AND MOLLER-EBERHARD

FIG. 3. Kinetics of arginine release from salmine. Arginine release was determined by amino acid analysis (0 - 0 - 0) or by the calorimetric assay (0 - 0 - 0). The linear portions of both curves extend beyond the initial 30 min of incubation which was selected for the assay.

residues per mol of salime. This result indicated that the salmine possesses four arginyl residues at the COOH-terminal end, a fact consistent with published sequence data for salmine and other protamines (13). Evidence that only arginine is released by SCPB is shown in Fig. 1. Amino acid analysis of the assay mixture incubated for various lengths of time shows that increasing amounts of arginine are released exclusive of other residues. Background amino acids are represented in this figure by glutamic acid, glycine, and valine, and all remained unchanged. These results indicate that no significant endopeptidase or aminopeptidase digestion occurs during the course of the incubation. Optimal Assay Conditions Figure 2 shows that the quantity of substrate selected for the assay (1 mg) is adequate for saturation of the enzyme. It has been previously reported

FIG. 4. Arginine release from salmine as a function of enzyme (serum) concentration. Fifty microliters of the I:4 diluted serum corresponds to the quantity used in the standard assay. The lower curve illustrates ninhydrin color development due to serum proteins in the TCA supematant.

SERUM

CARBOXYPEPTIDASE

I-, 2

I

, 6

8 10 12 ,,m (.InYt*sl

B (SCPB) ASSAY

I,

16

/ 18

47

26

FIG. 5. Time course of ninhydrin color development at 100°C due to free arginine. An incubation time of 7.5 min was selected for the standard assay.

that SCPB is a metalloenzyme and that addition of CoCl, enhances its activity (15,16). This enhancement of SCPB by CoCl, has been studied with respect to time and these results are given in Table 2. Incubation of serum for 2 hr with 5 mM CoCl, was chosen so that multiple assays could be completed in a 4 to 8 hr period. The quantitative validity of the results requires that the rate of arginine release must remain constant throughout the course of the assay. The kinetic analysis of arginine release is shown in Fig. 3 and indicates that during the initial 30 min, the interval selected as standard assay condition, arginine release is constant. The calorimetric analyses of arginine released by SCPB during the incubation was compared with amino acid analysis results and good agreement was obtained between the progression of the two curves, The relationship between the arginine released and the quantity of SCPB added to the reaction mixture was also examined. Figure 4 shows that a linear relationship exists over a wide range of SCPB concentrations. The quantity of serum used in the assay was 50 ~1 of I:4 diluted serum which produces a 0.17 to 0.23 absorbance difference after 30 min of incubation 0.6 1

FIG. 6. SCPB activity as a function of pH. Serum treated with 5 mM CoCl, for 2 hr at room temperature was assayed for SCPB activity in MES and HEPES buffers over the pH range of 5.5 to 8.8. The optimum was estimated to be at pH 6.8.

48

CORBIN,

HUGLI

AND MULLER-EBERHARD

FIG. 7. SCPB is detected in the cu-globulin fraction after separation of human serum on a Pevikon block by electrophoresis at pH 8.6. Protein was estimated by Lowry-Folin analysis (0 - 0 - 0). The SCPB activity was detected by the calorimetric assay (0 - 0 - 0).

for serum from normal individuals. The absorbance obtained from the assay mixture is also dependent on the length of time the sample is heated once the ninhydrin reagent is added. Figure 5 shows that the rate of ninhydrin color development for free arginine in the absence of serum is rapid. Background color development under assay conditions does not exceed 0.04 optical density units after 7.5 min of incubation. Since longer incubation with ninhydrin would result in a greater color contribution by TCA soluble serum proteins (5) 7.5 min was chosen as the standard condition for this assay. Examination of Fig. 5 shows that a variation of 30 set of incubation time would result in less than 5% variation in total absorbance. Dependence of SCPB activity on pH was examined and the result is shown in Fig. 6. It can be seen that pH 7.2, the condition selected for the assay, is approximately the pH optimum for the Coz+-activated enzyme. The pH of the assay mixture was determined at various intervals during the course of the incubation and no change was recorded. 700, 6OOj

sooi

6

t

100 i

i

lornal

FIG. 8. SCPB activity in normal and pathological sera. The mean value from 13 healthy sera was 395 units of SCPB activity. Means of 302 and 170 units were obtained for sera from patients with dextran shock and Dengue hemorrhagic fever. respectively. The mean values are designated by the horizontal lines.

SERUM

Detection

CARBOXYPEPTIDASE

B (SCPB) ASSAY

49

of SCPB in Serum Fractions

Normal human serum was fractionated by Pevikon block electrophoresis and by sucrose density gradient ultracentrifugation and the fractions were analyzed for protein content and SCPB activity. Following electrophoresis the SCPB activity was detected in the o-globulin region (Fig. 7). Ultracentrifugation of serum indicated the SCPB activity was associated with a protein zone sedimenting at a velocity of 11s. These results are in agreement with previously published data on purified SCPB (1). SCPB Activity

in Normal

and Pathological

Sera

SCPB activity of normal sera was compared to the activity of sera from patients with Dengue shock syndrome and from patients developing shock upon intravenous infusion of dextran. As shown in Fig. 8, the normal sera exhibited a range between 318 and 466 units of SCPB activity with the mean at 395 units. Patients in shock after dextran infusion exhibited subnormal SCPB activity and a significantly lowered mean of 302 units. Sera from patients with Dengue shock syndrome also had depressed SCPB activity values and an abnormal mean of 170 units. Determination of the total concentration of serum protein in patients and in normal sera confirmed that generally depressed protein levels were not responsible for the subnormal levels of SCPB activity. In addition, 10 sera from patients with cystic fibrosis were assayed and the SCPB activity in all of these sera were within the normal range. Eight of the sera were obtained from patients homozygous for cystic fibrosis and two of the patients were heterozygous. It was shown that no quantitative differences existed in SCPB activity of homozygous and heterozygous individuals. The median SCPB activity for all cystic fibrosis patients was identical to that of normal individuals with a range of 350 to 495 units. DISCUSSION

Human serum carboxypeptidase preferentially removes the basic COOH-terminal amino acid residues lysine and arginine from natural and synthetic peptide substrates. It has recently been shown that SCPB is more similar in specificity to a liver carboxypeptidase than to pancreatic carboxypeptidase B. Both serum and liver carboxypeptidases cleave COOH-terminal lysine faster than arginine, while the opposite applies to carboxypeptidase B (2). SCPB is reportedly a metalloenzyme and can be inhibited by addition of EDTA (15). Inactivation of SCPB by EDTA was, however, found to be incomplete in serum, since after 24 hr of incubation with 2 mM EDTA, approximately 5% of the initial enzyme activity remained. It is unknown whether this residual activity belongs to a fraction of native SCPB which retains the metal ion, to the apoenzyme

50

CORBIN.

HUGLI

AND MOLLER-EBERHARD

which expresses a lower level of activity, or to a second, minor enzyme which is distinct from the major SCPB (1). Evidence presented in this paper indicates that the procedure describes an assay for the 11s CYglobulin SCPB. Known properties of salmine indicated that this protamine could serve as a substrate for SCPB. The amino acid composition and sequence of salmine proved particularly advantageous in developing a calorimetric assay forquantitating SCPB activity. The NH,-terminal residue of salmine is proline which was shown to block digestion of salmine by serum aminopeptidase. The terminal prolyl residue makes a negligible contribution at 570 nm after reaction with ninhydrin. Absence of primary amino groups in salmine further minimizes the contribution by the intact substrate to the ninhydrin reaction. The repeating sequence of four arginines at the COOH-terminus of salmine proved particularly susceptible to hydrolysis by the carboxypeptidase B-like serum enzyme. Kinetic measurements showed that SCPB activity could be accurately measured calorimetrically with 10 to 20 ~1 of human serum. Other substrates such as benzyl-glycylarginine or hippurylarginine could substitute for salmine in the calorimetric assay, except that the reduced turnover with these substrates would necessitate using considerably longer incubation times or larger quantities of serum than those employed in the described procedure. The calorimetric assay may prove immediately valuable for the clinical investigator. A number of patients who developed shock following intravenous dextran infusion had somewhat lower SCPB levels than healthy individuals. The mean difference was approximately 25%. This difference is probably significant since the total protein concentrations of the patients’ sera were normal. All sera from patients with Dengue shock syndrome exhibited markedly depressed SCPB activity. Total serum protein was also reduced in some of these sera. Metabolic studies have shown that C3g turnover is enhanced and that the serum concentration of various complement proteins falls significantly during shock (9). Since rigid control of the complement derived anaphylatoxins is presumed essential for maintaining stable circulation, a 25-50% diminution in SCPB activity might contribute to development of a clinical shock syndrome. Evidence exists that marked changes in pressure and diameter of arterioles are produced by topical applications of ng quantities of the C3a anaphylatoxin (17). A genetic deficiency of SCPB has been postulated to occur in patients with cystic fibrosis accounting for expression of a factor in serum from cystic fibrosis homozygotes or heterozygotes which induces ciliary dyskinesis (18). We therefore examined a number of sera from cystic 9 The third component of the blood complement system.

SERUM

CARBOXYPEPTIDASE

B (SCPB) ASSAY

51

fibrosis patients and found all to be normal with respect to SCPB activity. These results refute the hypothesis that a deficiency in SCPB might be the primary gene defect in cystic fibrosis (19). Values for SCPB activity in the sera of cystic fibrosis patients have recently been reported by Lieberman (20). Our results agree with these findings. REFERENCES 1. Bokisch, V. A., and Mtiller-Eberhard. H. J. (1970) J. C/in. Invest. 49, 2427, 2. Oshima, G.. Kato, J., and Erdos, E. G. (1974) Biochim. Biophys. Acra 365, 344. 3. Folk, J. E., Piez, R. A., Carroll, W. R., and Gladner. J. A. (1960) J. Biol. Chem. 235: 2272.

Yemm, E. W.. and Cocking, E. C. (1955)AnaI~st 80, 209. 5. Matheson, A. T., and Tattrie, B. L. (1964) Canad. J. Biochem. 42, 95. 6. Bokisch, V. A., Mtiller-Eberhard, H. J., and Dixon. F. J. (1973) Trans. Assoc. 4.

Physicians

Amer.

86, 102.

7. Johnson, U., and Laurell, A. B. (1974) Scand. J. Itnmunol. 3, 673. 8. Bulletin, World Health Organization (1973) 48, 117. Report of an international collaborative study. 9. Bokisch. V. A., Top, F. H.. Jr.. Russell, P. K.. Dixon, F. J.. and Mtiller-Eberhard. H. J. (1973) New England J. Med. 289, 996. 10. Spackman. D. H., Stein, W. H.. and Moore, S. (1958) Anal. Chem. 30, 1190. Il. Spackman, D. H. (1963) Fed. Proc. 22, 244. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)~. Bid. Chem. 193, 265.

13. Ando, T., and Watanabe. S. (1969) Int. J. Prof. Res. 1, 221. 14. Edman, P., and Begg. G. (1967) Eur. J. B&hem. 1, 80. 15. ErdBs, E. G., Sloane, E. M., and Wohler. 1. M. (1964) Biochem.

Phnrmacol.

13,

893.

16. Erdos, E. G., Yang, Y. T.. Tague, L. L., and Manning, N. (1967) Biochem.

Pharmacol.

16, 1287.

17. Mahler, F., Intaglietta. M., Hugh, T. E., and Johnson, A. R. (1975) Microtwsc.Res. 9, 345.

Conover, J. H.. Conod, E. J., and Hirschhorn, K. (1973) Lancer 2, 1501. 19. Conover, J. H., Conod, E. J.. and Hirschhorn, K. (1974) L[fe Sci. 14, 253. 20. Lieberman, J. (1975) Amer. Rev. Resp. Dis. 111, 100. 18.