Mode of Action of Arboxypeptidase

8/11/2019 Mode of Action of Arboxypeptidase

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CARBOXYPEPTIDASE B

II . MODE OF ACTION ON PROTEIN SUBSTRATESAND ITS APPLICATION TO CARBOXYL

TERMINAL GROUP ANALYSIS*

BY JULES A. GLADNER AND J. E. FOLKt

(From the National Instilute of Dental Research and the National Institute of Arthritisand Afetabolic Diseaaea, National Inatitutes of Health, Public Health Service,

United States Department f Health, Education, and Welfare, Bethesda, Maryland)(Received for publication, August 19, 1957)

In a previous publication 1) the isolation, partial purification, and eluci-dation of the specificity of carboxypeptidase B were described. The en-zyme was shown to hydrolyze lysine, arginine, ornithine, and homoargininefrom the C-terminal position of synthetic peptide substrates.

In recent years carboxypeptidase A2 has been widely used for the identi-fication of C-terminal amino acid residues and partial sequence analysisof proteins and peptides 2). Carboxypeptidase A was shown to actslowly, if at all, upon basic amino acid residues 3, 4), and, therefore, theidentification of these amino acids was in question. Since carboxypepti-dase B rapidly hydrolyzes lysine and arginine bonds (1)) an investigationof the action of this enzyme upon a number of protein and peptide sub-strates was undertaken. This report describes the release of basic aminoacids by carboxypeptidase B upon its reaction with protein substrates,singly and in conjunction with carboxypeptidase A.

Mabrials

Carboxypeptidase B was prepared as previously described 1) with oneof the two following modifications.

Method I-An aliquot of 2.5 ml. of final stock solution of procarboxypep-tidase B containing 4 to 5 mg. of protein per ml. was adjusted to pH 8.0

* Presented in part before the 131st meeting of the American Chemical Society,Miami, Florida, April 7-12, 1957.Presented in part before the Forty-eighth annual

meeting of the American Society of Biological Chemists, Chicago, Illinois, April15-19,1957.

t Research Associate, American Dental Association.1 The following abbreviations will be employed throughout this paper: C-terminal

and N-terminal for the carboxyl terminal and amino terminal amino acid residues ofproteins and polypeptides, DFP for diisopropy1 phosphorofluoridate, DIP for diiso-propylphosphoryl, S:E for the molar ratio of substrate to enzyme, DNP for dinitro-phenyl.

* For clarity of nomenclature Ansons classical carboxypeptidase will be referredto as carboxypeptidase A.

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394 CARBOXYPEPTIDABE B. II

to 8.3 by the addition of 10 mg. of KsHP04. Trypsin (6.25 mg. in 2.5

ml. of 10e3 M HCl) was added, and the mixture was placed in a dialysissack and dialyzed with stirring against large quantities of 0.2 M NaCl,pH 8.0, at 25. The dialysis was st,opped after 1 hour. Tryptic activa-tion was terminated by the addition of a 200-fold molar excess of DFP,and the activated mixture was then made up to a total of 10 ml. with 0.2MNaCl. AIethod II was similar to Method I with t,he exception that dialysiswas omitted. After 1 hour of incubation with trypsin, the activat,ed solu-tion was stirred with 600 mg. of a mixture of ion exchange resins (1: 2 wet

weight mixture of Dowex 5OW-X8 (H form) and Dowex 2-X10 (OH form),both 50 t,o 100 mesh) for 20 to 30 seconds. Following removal of the ionexchange resins, the pH was quickly readjusted to 8.0, DFP was added(pH maintained at S.O), and the volume was adjusted as in Method I.Method II yielded an extremely low enzyme blank. 0.5 ml. of theseenzyme solutions was used per Imole of protein substrate after routineesterase assays for trypsin (5) showed no traces of residual tryptic activity(20 to 30 minut.es after addition of DFP). In all experiments, prepara-tions of carboxypeptidase B, starting from acetone powders, were made upand used on t,he same day. In t,he preliminary investigat,ions reported inthis paper no attempt was made to discern maximal and minimal concen-trations of enzyme applicable to the method. Such data will be reportedin a future publication.

S&nine-Two preparat,ions were employed. One of them was kindlysupplied by Dr. L. Weil of the Eastern Utilization Branch of the UnitedStates Department, of Agriculture (Preparation I). The other was pre-pared in t,his laboratory (6), extreme care being taken during the prepara-tion to avoid hydrolysis (Preparation II). Bot,h protamines were in thesulfate form. Preparation II was also used as a conbrol to ascertain thepotency of t,he individual carboxypeptidase B preparations.

DIP-u-chymotrypsin was prepared by employing 0.2 M P-phenylpropi-onic acid by a method essentially the same as that reported by Dreyerand Neurath (7).9

Chymotrypsinogen was obtained from the Worthington Biochemical Cor-poration, Freehold, New Jersey, as the damp filter cake of first crystals.

It was recrystallized six t,imes from ammonium sulfate, dialyzed salt-freeagainst 1O-3 fii HCl at 4, and lyophilized.

Trypsin and DIP-Trypsin-Four lots of Worthington crystalline trypsinwere used (Lots TL-581,433-C, 436, and TR-20-SF). They were all dialyzedsalt-free and lyophilized when required. The salt-free enzyme powder(10 mg. per ml.) was dissolved in cold (0) 0.05M tris(hydroxymethyl)-

3 We m-ish to t.hank Dr. W. J. Dreyer for his assistance during the course of thispreparation.

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J. A. GLADSER ASD J. E. FOLK 395

aminomethane buffer, pH 8.0. Immediately thereafter a 200-fold molar

excess of DFP in isopropanol was added, the pH being maintained between7.8 and 8.0. Esterase determinations for t.rypt.ic activit,y showed essen-tially complet,e inactivat,ion in about 20 to 30 minutes. The solutionswere quickly adjusted to pH 3.0 and dialyzed against large quantities oflea M HCl at 4.

Another preparation of DFP-inhibited t,rypsin from Lot TL-581 wasmade under the exact conditions described above with the exception thatthe solut,ion was made 0.10 M in CaC and 0.10 M in fi-phenylpropionic

acid. It was designated as Lot TL-581-Ca.Trypsinogen Activation MixturesTrypsinogen, Worthington Lot Tg-521, was completely activated under conditions reported by Neurath et al.(8); activity and change in optical rotation were in accordance with thereport,ed data. In anot,her study, 0.1 M @-phenylpropionic acid, a com-petitive inhibitor of chymotrypsin (3), was used to suppress possible hy-drolysis by chymotrypsin. In all activation experiments, at time intervalsof 1, 2, 3, and 24 hours, aliquots were withdrawn, and DFP in 200-foldmolar excess was added t,o terminat,e the reaction, the mixtures being keptat 0. Loss of activity was followed by esterase assays. After completeDFP inact,ivation t.he pH was lowered to 3, and the contents were centri-fuged when required, followed by dialysis against 10-a M HCl at 4.

Carboxypeptidase A-Starting materials were Worthington, three timescrystallized, Lots CO-561, CO-568, and CO-569. All preparations con-tained varying degrees of carboxypeptidase B activity when assayedagainst hippuryl-L-arginine. Subsequent recrystallizations by the gradientdialysis techniques and isoelectric crystallizations as described by Neurath,Elkins, and Kaufman (9), and modified by heurath and Schwert (cf. (3)),removed all but traces of this activity.

Protein concentrations in these experiments were determined spectro-phot.ometrically; extinct,ion coe6icient.s were l&2 = 20.6 for chymotryp-sinogen (lo), EZ~% = 14.4 for trypsin, E , = 13.9 for t,rypsinogen (11).Carboxypeptidase A concentrations were determined at 278 rnp, assuminga molar extinction coefficient of 8.6 X lo4 (12).

Light Meromyosin-These crystalline preparations, prepared by the shortterm action of trypsin on myosin (13), were kindly supplied by Dr. K.Laki of this laboratory.

Performic acid-oxidized chick globin was kindly supplied by Dr. JeanRotherham of the National Heart Institute, National Institutes of Health.The dialyzed and lyophilized salt-free preparation was completely solublein distilled water at pH 8.2.

Poly-r,-lysineO HBr (46 mer) was synthesized in this laboratory (14).

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396 CARBOXYPEPTIDASE B. II

EXPERIMENTAL

Carboxypeptidase B preparations and substrates, in amounts previouslyspecified, were incubated for various time periods under direct pH controlat 25 and pH 8.0 to 8.2. In certain experiments carboxypeptidase A,S:E = 25: 1 prepared as previously reported for end group analysis (4)was added at specific times. When protein substrates were DIP enzymes,a 50-fold molar excess of DFP was added to the substrate solution priorto adjusting the pH to 8.0. Esterase assays against their specific syn-thetic substrates (3) showed no traces of active enzymes after such treat-

ment.The reactions were terminated by adding aliquots directly to an excess

of Dowex 50-X12 (H form). The mixtures were then treated, and theamino acids were eluted from the resin as previously described (4). Incertain experiments, where indicated, the mixtures were dinitrophenylated,and the amino acids were identified and estimated according to the methodof Fraenkel-Conrat et al. (2).

Initial identifications of a portion of the ion exchange resin eluents were

carried out on two-dimensional paper chromatograms by employing thetechnique of Irreverre and Martin (15). The chromatograms, run onSchleicher and Schuell No. 598 filter paper, were lightly sprayed on oneside with a sensitive Sakaguchi reagent for arginine.4 Following this pro-cedure the entire paper was dipped in 0.25 per cent ninhydrin in acetonecontaining 5 per cent (v/v) glacial acetic acid for identification of otheramino acids.

For quantitative purposes, arginine was determined by a calorimetric

Sakaguchi procedure developed in this laboratory by Hayden and Irre-verre.6 When necessary lysine and other amino acids were determinedquantitatively by the ion exchange method of Moore and Stein (16) orthe DNP technique of Fraenkel-Conrat et al. (2).

Results

SaZmine-Table I shows the results obtained upon the incubation ofcarboxypeptidase B preparations, of equal potency6 against syntheticsubstrates, with two preparations of salmine sulfate. The results of theaddition of carboxypeptidase A after 2 hours prior incubation with car-boxypeptidase B are also illustrated.

Preparation II, in later stages of these investigations, was employedto estimate enzyme potency against protein substrates. For compari-

4 Irreverre, F., Kominz, D., and Hayden, A., to be published.6 Hayden, A., and Irreverre, F., to be published.6 Units of carboxypeptidsse B are defined as 20 X Ko, where Ko = the zero order

rate constant with 0.025 M hippuryk-arginine as substrate.

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J. A. GLADNER AND J. E. FOLK 399

A purified preparation of carboxypeptidase B yielded essentially the same

results as were obtained with the supernatant materials. Amino acidanalyses were performed by the DIW technique of Fraenkel-Conrat et al.(2).

Preliminary experiments with carboxypeptidase B were carried out onlight meromyosin. In all cases the only major amino acid released bycarboxypeptidase B preparations was lysine. Poly-n-lysine, when in-cubated with carboxypeptidase B, yields only free lysine. Quantitativeestimations were not performed.

Table IV summarizes some of the data pertaining to the effect of car-boxypeptidase B on various protein substrates.

TABLE IVAmino Acids Liberated from Protein Substrates by Carboxypeptidase A

and Carboxypeptidase B

Substrate

ChymotrypsinogenTrypsinogenCommercial trypsinActivated trypsinogen*DIP-r-chymotrypsinSalminePerformic acid-oxidized chick globinLight meromyosinPoly-L-lysine (46 mer)

-

-

Carboxypeptidase B

0

0

Lysine0Arginine

ILysine

I

-

-

Carboxypeptidase A

0

0

0

0

0

0

Histidine00

* Trypsin-activated, see the text for the details.

DISCUSSION

The present experimental data extend to protein substrates our knowl-edge of the specificity of carboxypeptidase B. It is evident that carboxy-peptidase B may be employed for the determination of basic C-terminalamino acid residues n proteins and peptides. With the consideration inmind of the limitations of other enzymatic methods of end group analysis,one may employ carboxypeptidase B, either alone or coupled with car-boxypeptidase A, to elucidate C-terminal amino acid patterns of certainproteins and peptides.

The inability of carboxypeptidase B to liberate free basic amino acidsfrom trypsinogen or chymotrypsinogen, coupled with negative results inprevious attempts to obtain C-terminal residues via carboxypeptidase A4, 17) or chemical techniques 18), demonstrates either that these zymo-

7 Estimated from the intensity of the ninhydrin color to be about 1 equivalent.

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400 CARBOXYPEPTIDASE B. II

gens contain C-terminal amino acids which are not identifiable by thepresent methods of C-terminal group analysis or that they contain no freea-carboxyl grouping.

Since the initial reports of a C-terminal lysine in DIP-trypsin (19) werein conflict with the negative results reported by chemical methods (18), amore complete study in which carboxypeptidase B was employed was un-dertaken. The experimental results indicate that many preparations ofcommercial trypsin, although crystalline, are chemically heterogeneous.These data support the findings that most of the tryptic activity remains

intact during the initial stages of autolysis (20) and suggest that perhapsthe trypsin molecule is held intact by chemical cross-linkages during theinitial stages of autolysis. On the other hand, trypsin resulting from a morerapid activation of trypsinogen contains virtually no lysine available tocarboxypeptidase B. This is compatible with the hypothesis of Davie andNeurath that the hydrolysis of the . . . ys . soleu bond in trypsinogencoupled with release of hexapeptide is sufficient to impart enzymatic ac-tivity (11).

The C-terminal sequence of amino acids of DIP+chymotrypsin hereto-fore inferred to be . . . leu.ser .arg (7) has now been verified through theuse of carboxypeptidase B coupled with carboxypeptidase A. Since thebest preparations of carboxypeptidase B to date contain a minimum of 4per cent carboxypeptidase A contamination (l), the appearance of tracequantities of other amino acids is probably attributable to the latter en-zyme. The appearance of small quantities of leucine following incubationof DIP+chymotrypsin with carboxypeptidase A is indicative of small

quantities of &chymotrypsin impurity (7).The results achieved with the performic acid-oxidized chick globin con-firm and extend to protein substrates the previous observation that C-termi-nal histidine is not removed by carboxypeptidase B (1).

The few examples of C-terminal group analysis presented here as real-ized by carboxypeptidase B are only representative examples of the poten-tial value of this enzyme in protein and peptide structure studies.* Theuse of carboxypeptidase B in conjunction with other methods opens the

way to C-terminal sequence analysis of peptides obtained during trypticdigestion of protein material as exemplified by elucidation of the C-termi-nal sequence of DIP-x-chymotrypsin.

8 One significant side light of the specificity of carboxypeptidase B is the releaseof S-(,%aminoethyl)cyeteine from a tryptic digest of beef insulin which had beenreduced and allowed to react with fl-bromoethylamine as outlined by Lindley (21).Tietze et al. (22).

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J. A. GLADNER AND J. E. FOLK 4701

SUMMARY

The specificity requirements of a new proteolytic enzyme, carboxypep-tidase B, have been examined by employing protein substrates. The en-zyme hydrolyzes lysine and arginine, but not histidine, from the carboxylterminal position of polypeptide chains. It can therefore be used to de-termine carboxyl terminal lysyl and arginyl residues in proteins and poly-peptides.

The authors wish to acknowledge the technical assistance of Miss Emilie

N. Smith. Our thanks are also due to Dr. K. Laki for his valuable dis-cussions during the course of this work.

BIBLIOGRAPRY

1. Folk, J. E., and Gladner, J. A., J. Biol. Chem., 231, 379 (1958).2. Fraenkel-Conrat, H., Harris, I. J., and Levy, A. L., in Glick, D., Methods of

biochemical analysis, New York, 2 (1955).3. Neurath, H., and Schwert, G. W., Chem. Rev., 46, 69 (1950).4. Gladner, J. A., and Neurath, H., J. BioZ. Chem., 206, 345 (1953).

5. Schwert, G. W., Neurath, H., Kaufman, S., and Snoke, J. E., J. BioZ. Chem.,173, 221 (1948).6. Callanan, M. J., Carroll, W. R., and Mitchell, E. R., J. Biol. Chem., 229,279 (1957).7. Dreyer, W. J., and Neurath, H., J. Biol. Chem., 2l7.527 (1955).8. Neurath, H., Rupley, J. A., and Dreyer, W. J., Arch. Biochem. and Biophys., 66,

243 (1956).9. Neurath, H., Elkins, E., and Kaufman, S., J. Biol. C&m., 170, 221 (1947).

10. Schwert, G. W., J. Biol. Chem., 190, 799 (1951).11. Davie, E. W., and Neurath, H., J. BioZ. Chem., 212, 507 (1955).12. Albrecht, G. S., cited in Vallee, B. L., and Neurath, H., J. BioZ. Chem., 217,253

(1955).13. Mih&lyi, E., and Saent-Gyiirgyi, A. G., J. BioZ. Chem., 201, 189 (1953).14. Folk, J. E., Arch. Biochem. and Biophys., 64, 6 (1956).15. Irreverre, F., and Martin, W., Anal. Chem., 26, 257 (1954).16. Moore, S., and Stein, W. H., J. BioZ. Chem., 192, 663 (1951).17. Davie, E. W., and Neurath, H., J. BioZ. C&m., 212,515 (1955).18. Niu, C.-I., and Fraenkel-Conrat, H., J. Am. Chem. Sot., 77,5882 (1955).19. Gladner, J. A., and Laki, K., in Folk, J. E., J. Am. Chem. Sot., 76, 3541 (1956).20. Nord, F. F., Bier, M., and Terminiello, L., Arch. Biochem. and Biophys., 66, 120

(1956).

21. Lindley, H., Nature, 176, 647 (1956); J. Am. Chem. Sot., 77,4927 (1955).22. Tietae, F., Gladner, J. A., and Folk, J. E., Biochim. et biophys. acta, 26,659 (1957).

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Mode of Action of Arboxypeptidase

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