32 ACTIVATION OF TRYPSINOGEN MECHANISM OF ACTIVATION OF TRYPSINOGEN AND CHYMOTRYPSINOGEN BY HANS NEURATH AND WILLIAM J. DREYER Dept. of Biochemistry, University of Washington, Seattle 5, Washington, U.S.A. Received 6th June, 1955 In an attempt to elucidate the mechanism of the tryptic activation of trypsinogen and chymotrypsinogen, respectively, the molecular changes occurring during the conversion of these zymogens to their respective active forms have been determined by physical and chemical means. These have included the methods of peptide and end-group analysis, electrophoresis and sedimentation, and enzymatic activity toward synthetic substrates. The tryptic activation of trypsinogen appears to be a one-step process accompanied by the release of an acidic hexapeptide from the N-terminal portion of the molecule.In contrast, the activation of chymotrypsinogen involves several enzymatically active inter- mediates, which have been characterized by end-group analysis and electrophoresis, and the release of a basic dipeptide, serylarginine. The active enzymes, unlike the zymogens, are subject to reversible dimerization. It is concluded that the primary chemical event requisite for activation is the hydrolysis of a peptide bond between a basic amino acid residue (lysine in the case of trypsinogen, and arginine in the case of chymotrypsinogen) and an isoleucylvaline sequence. It remains to be determined whether concomitant intramolecular rearrangements are also involved in the transformation of zymogens to the active form. It is a well-established fact, recognized by physiologists and chemists many decades ago, that the proteolytic enzymes of the digestive tract are secreted by the cells in an inactive form and through extracellular transformations become active enzymes, capable of hydrolyzing proteins and peptides.These transforma- tions of zymogens to the active catalysts present features of unusual interest : 1 9 2 they are enzyme catalyzed and it has not yet been possible to duplicate these transformations by nonenzymatic means. The activating enzymes are proteolytic enzymes themselves which operate with a high degree of specificity and selectivity. In all known cases is the conversion of zymogen to the active form an irreversible process. The activation process represents the final steps in the formation of aH .NEURATH AND W. J . DREYER 33 biologically active protein and thus offers a close view of the relation of chemical structure to biological activity of the proteins. As the nature of the changes occurring during activation becomes recognized, inferences may be drawn of the nature of the catalytically active centre. Since it seems now well-established that both chymotrypsin and trypsin have only one active centre per molecule,3 the question arises whether the structural configuration characteristic of the centre is preexistent in the zymogen molecule or whether it is created during activation by a process of intramolecular rearrangement. Tine present discussion will deal with two activation systems, i.e., trypsinogen and chymotrypsinogen ; some of the previously reported findings 4.5969 7 will be integrated with data which have been more recently obtained in this laboratory and elsewhere.Since during enzymatic activation of the zymogens peptide bonds are opened, new terminal groups are formed. Although no large molecular weight changes occur, the release of small peptide fragments may cause changes in the electrical charge of the protein components. On the basis of these considerations, the following experimental methods were employed conjointly in the study of the activation process : (i) end-group analysis, employing Sanger's 1 : 2 : 4-fluorodinitrobenzene (FDNB) reagent for amino terminal groups and carboxypeptidase 49 9 for carboxyl terminal groups; (ii) peptide isolation by paper or ion exchange chromatography or by paper electrophoresis, followed by amino acid and end-group analysis of the isolated peptides ; (iii) electrophoretic analysis of the protein components of the activation mixture; (iv) sedi- mentation analysis in the ultracentrifuge ; and (v) measurement of enzymatic activity with the use of synthetic substrates (benzoyl-L-arginine ethyl ester for trypsin, acetyl-L-tyrosine ethyl ester for chymotrypsin).THE ACTIVATION OF TRYPSINOGEN Of the two activating systems considered herein the conversion of trypsinogen to trypsin will be considered first since, apparently, it presents a one-step reaction which does not involve the formation of intermediate products. The activation of trypsinogen can be independently catalyzed by three proteolytic enzymes,l i.e.(i) enterokinase, (ii) trypsin, and (iii) penicillium kinase. While the first of these is probably physiologically the most important, the autocatalytic conversion under the influence of trypsin has the operational advantage that no new protein is introduced into the system. The activation by penicillium kinase has the practical merit that the reaction occurs at an acid pH (PH 3) where autolytic degradation of trypsin is held to a minimum. Maximum autocatalytic conversion of trypsinogen to trypsin at pH 7.8-8 requires the presence of calcium ions to suppress the formation of enzymatically " inert protein ". From a kinetic view- point, the reaction can be formulated in terms of a bimolecular product-catalyzed chemical reaction.1 The molecular-kinetic properties of trypsinogen 10 and trypsin 11 have been considered at a previous Discussion of the Faraday Society.12 Some of the chemical and physical properties of trypsinogen and trypsin (or DIP-trypsin)," pertinent to considerations of the present problems, are summarized in table 1.Within the limits of the experimental error, the zymogen and the active enzyme have identical molecular weights of approximately 23,800, this value being in good agreement with the minimum molecular weight determined from amino acid analysis 13 (see below). Although no significant change in molecular weight accom- panies the tryptic activation of trypsinogen, the data summarized in table 1 clearly point toward the liberation of an acidic peptide from the N-terminal sequence * Here and elsewhere, DIP refers to di-isopropylphosphoryl, DFP to di-isopropyl- phosphofluoridate, and TCA to trichloracetic acid.B34 ACTIVATION OF TRYPSINOGEN of the trypsinogen molecule. Thus, according to the work of Desnuelles and co-workers, the N-terminal valine of the single polypeptide chain of trypsinogen is replaced by an isoleucine group, and the iso-ionic point of the protein shifts toward more alkaline regions. It is of interest that even after denaturation by 6 M urea, both proteins remain unreactive toward carboxypeptidase? As shown by representative data in table 2, the amino acid composition of trypsinogen and DIP-trypsin is almost identical. The observed differences in aspartic acid and lysine are in accord with the composition of the activation peptide (see below) and the calculated minimum molecular weights agree favourably with those determined by physical methods. TABLE 1 .-SOME PHYSICAL AND CHEMICAL PROPERTIES OF TRYPSINOGEN AND TRYPSIN (OR DIP TRYPSIN) trypsinogen trypsin molecular weight (sedimentation, diffusion) 23,800 10 23,800 11 E;g 13.9 14.4 N-terminal5 1 valine 1 isoleucine C-terminal (carboxypeptidase) 7 none none iso-ionic point (mixed bed ion exchange resin) 9.3 10.1 TABLE 2.-sOME COMPARATIVE DATA OF THE AMINO ACID COMPOSITION* OF TRYPSINOGEN AND DIP-TRYPSIN 13 amino acid aspartic acid glutamic acid arginine lysine histidine phenylalanine tyrosine proline 112 cystine trypsinogen 25.4 11.0 2.0 14-5 3.1 4.0 9.4 8.0 6.5 total N minimum molecular weight (calculated) 23,320 f 280 * given in terms of amino acid residue per mole.j- determined as cysteic acid. DIP-try pin 21-5 11.1 2-0 14.0 3.0 4.1 9.9 8.0 - 16.7 % 23,020 f 340 The activation peptide has been isolated and identified. In these experiments aliquots were removed from activation mixtures at various stages of activation, free amino acids and peptides adsorbed on to and eluted from Dowex-50 ion- exchange resin and separated on Dowex-50 ion-exchange columns (2 % cross- linked, sodium form). A representative chromatogram is shown in fig. 1. The amount of the peptide, represented by the major peak, was found to be proportional to the extent of activation, whereas the other, minor peaks failed to reveal any quantitative relation to the appearance of enzymatic activity and may, therefore, be ascribed to non-specific autolysis of the proteins.Quantitative amino acid analysis of the isolated peptide fraction yielded valine, aspartic acid and lysine in mole ratios of 1 : 4 : 1 in addition to fractional amounts of ammonia, probably introduced during the operation. The N-terminal position of valine was verified by Sanger's FDNB technique 8 and no other ether soluble DNP amino acid could be found; and since no valine appeared in the aqueous layer, the presence of only one valine per peptide was demonstrated. (E-DNP lysine was the only detectable DNP amino acid in the aqueous layer.) The C-terminal position of lysine was inferred from the specificity requirements of the activating enzyme, trypsin, but could not be demonstrated experimentally since the isolated peptide was unreactive toward carboxypeptidase.H .NEURATH AND W. J . DREYER 35 Accordingly, the structure val-(asp)4-lys has been assigned to this hexapeptide. The predominantly acidic properties, resulting from the presence of four aspartic acid residues, are in accord with the alkaline shift in iso-ionic point of trypsinogen during activation and was further confirmed by paper electrophoresis. In the presence of a citrate buffer, ionic strength 0.05, the isoelectric point was found to be 34-35, which is within the range expected for a polyvalent compound of the proposed structure. It is of interest to note that the peptide had no inhibitory effect on the esterase activity of trypsin when the enzyme was incubated with a 25-fold molar excess of the peptide.0 . 5 0 0 5 ACTIVATION PEPTIDE 0.400 0.300 0.200 0 100 I- pH 4.0 BUFFER I-PH 5.0 0.300- 0.200- 0.100- pH 5.0 BUFFER pH 6.0 BUFFER- ml. EFFLUENT FIG. 1.-Chromatogram (Dowex-50 ion exchange resin) of the peptide fraction of an activated sample of trypsinogen.7 I I Val - (Asp),-Lys Ileu ' 7 2 ; Trypsinogen + Val - (Asp), - L y s + Ileu '-c7 Peptide Trypsin FIG. 2.-Schematic representation of the tryptic activation of trypsinogen. The absence of a C-terminal amino acid, reactive toward carboxypeptidase, is indicated by the looped chains of the trypshogen and DIP-trypsh molecules. On the basis of all of these findings, the autocatalytic activation of trypsinogen may be depicted as shown in fig. 2. According to this scheme the single autolytic event accompanying activation is the splitting of the lysine-isoleucine bond, giving rise to the hexapeptide and the protein with an N-terminal, isoleucyl-valine sequence.* The general significance of these findings in relation to the mechanism of activation will be considered more fully in the discussi0n.t * The resulting decrease in molecular weight (705) is in accord with the change in ex- tinction coefficient (table 1).-f It is important to recognize that the experiments just described do not exclude the possibility that other peptides may have been liberated as well. All that can be said at this time is that no other ninhydrin-reactive peptide has yet been found which bears any quantitative relation to the amount of trypsin formed and which is subject to quantitative elution from Dowex-50 ion exchange resin.36 ACTIVATION OF TRYPSINOGEN THE ACTIVATION OF CHYMOTRYPSINOGEN The activation of this zymogen represents a more complex process since it is catalyzed by two enzymes, trypsin and chymotrypsin, and involves several en- zymatically active intermediates.The nature of the products of activation depends, therefore, on the concentration of both chymotrypsinogen and trypsin, a phenomenon which was clearly recognized by Jacobsen 14 by kinetic studies of the activation process. Under conditions of " rapid " activation (chymotryp- sinogen : trypsin ratio approximately 30 : 1) 6-chymotrypsin is the major end- product, whereas under conditions of " slow " activation (chymotrypsinogen : trypsin ratio 10,000: 1) the proteins described by Kunitz as a, and y-chymo- trypsins seem to be the major products of the reaction.Much of the published work on chymotrypsin was carried out with the crystalline product described by Kunitz 1 (a-chymotrypsin) without the realization that it represents electro- phoretically a heterogeneous mixture (see below). For this reason it is difficult ACTIVATION TIM E ELECTROPHORESIS 3.3 min. 6.0 min. 2 0 min. 55 min. 90 min. 1262 min. A 1389 min. I017 min. 1723 min. CHTG. 7- 8- FIG. 3.-Electrophoretic diagrams (ascending) of rapid activation mixtures. The patterns have been aligned horizontally to facilitate comparison of the three electro- phoretic components, chymotrypsinogen 7r and 8 chymotrypsins.23 to infer from a comparison of the chemical and physical properties of chymotryp- sinogen and a-chymotrypsin the nature of the changes which occur during the activation process.It is clear, however, that the two proteins have similar molecular weights 15~16 and that during the process several peptide bonds are opened ; thus, during the conversion to a-chymotrypsin four new end-groups are formed, i.e., N-terminal isoleucine and alanine,l7 and C-terminal leucine and tyrosine.9 Chymotrypsinogen is a homogeneous protein, as judged by solubility,l sedimentation,*s ion-exchange chromatography 18 and electrophoresis. Accord- ing to recent analytical data,lg the zymogen contains 18 basic amino acids (12 lysines, 2 histidines and 4 arginines) but only 12 acidic groups; there are 8 half- cystine residues (assumed molecular weight of 23,000).The protein is devoid of any reactive C-terminal groups 9 but contains one N-terminal half cystine.20H . NEURATH A N D W. J . DREYER 37 Rapid activation of chymotrypsinogen (chymotrypsinogen, 40 mg/d ; trypsin, 1.2 mg/ml, sodium phosphate buffer pH 7.8,0.05 M, 0") is accompanied by changes in electrophoretic patterns and mobilities and by the appearance of terminal groups. Characteristic electrophoretic patterns obtained in the presence of DFP (acetate buffer pH 4.98, ionic strength 0.1) are shown in fig. 3. It will be noted that during activation, the boundary corresponding to chymotrypsinogen is replaced by components of decreasing electrophoretic mobility. The mobility differences between chymotrypsinogen and the first activation product, rrchymo- trypsin are so small6 that they require prolonged electrophoresis for a visible resolution of the components.?-r-Chymotrypsin has been obtained in nearly pure form, as judged by electrophoresis, if activation was carried out in the presence of 8-phenylpropionic acid. Much larger mobility changes occur when rapid activation is permitted to proceed to the formation of 8-chymotrypsin. Evidence has already been presented to show that the conversion of rr-chymotrypsin to the &form is catalyzed by chymotrypsin rather than by trypsin.6 Thus the appear- ance of the electrophoretic peak characteristic of 8-chymotrypsin is greatly retarded by the addition of 8-phenylpropionate, an effective inhibitor of chymotrypsin, even though the activity of the activation mixtures remained unchanged.In contrast, soybean trypsin inhibitor had no effect on the rate of formation of 8-chy mo tr ypsin. The protein components resulting from rapid activation have been character- ized by end-group analysis; the results are summarized in table 3. Thus T- TABLE 3 .-DISTRIBUTION OF TERMINAL GROUPS * IN CHYMOTRYPSINOGEN, ACTIVATION MIXTURES AND CRYSTALLINE CHYMOTRYPSINS protein activation N-terminal C-terminal - CYSP chymotrypsinogen 20 - n-chymotrypsin 6 rapid cys/2 iIeu-vaI (a%> -1- 8-chymotrypsin 6 . 2 1 , * 2 rapid cys/2 ileu-Val leu chymotrypsin 6 extended, rapid cys/2 ileu-val leu tyr a-chymotrypsin 6 . 9 , 17 slow cys/2 ileu-Val ala leu tyr ,f3 : y-chymotrypsin 9,17 extended, slow (cys/2?) ileu-Val ala leu tyr * N-terminal groups were determined as DNP-derivatives, C-terminal groups with t inferred from composition of activation peptide (see below).the use of carboxypeptidase. chymotrypsin differs from chymotrypsinogen by the presence of an additional N-terminal group, i.e. isoleucine, whereas a-chymotrypsin possesses, in addition, a C-terminal leucine group. All three proteins contain the N-terminal half- cystine group. While rr-chymotrypsin has not yet been obtained in a crystalline form, the crystallization of a protein from an activation mixture corresponding to 6-chymotrypsin has been accomplished after inactivation with DFP.6s 22 However, electrophoretic analysis showed that no purification was achieved by this procedure; 6 on the contrary a solution of the crystals appeared electro- phoretically more heterogeneous than the original activation mixture. The appearance of a C-terminal leucine residue, together with a large shift in electrophoretic mobility during the T-8 conversion,6.23 provided presumptive evidence for the liberation of a basic peptide, and hence, attempts were made to determine the presence of this peptide, to isolate it, and to determine its chemical structure .23 To this end, activation mixtures were prepared and when the formation of 8-chymotrypsin was complete, the reaction was stopped by the addition of DFP.The fraction soluble in 10 % TCA was subjected to column chromatography on XE-64 ion-exchange resin. A representative chromatogram is shown in fig. 4. The first major peak appearing was found to consist of a mixture of acidic and neutral peptides which were subsequently resolved on a Dowex-50 column in the38 ACTIVATION OF TRYPSINOGEN sodium cycle.These are shown in the lower half of fig. 4. These peptides, none of which occurred in stoichiometrically significant quantities, have probably arisen from secondary proteolytic degradations. The centre peak was found to be primarily due to ammonia whereas the peak on the right represents the activation peptide. It appears at the same effluent volume as free arginine and, upon hydro- lysis, was found to yield only serine and arginine in a mole ratio of approximately 1 : 1. The presence of arginine was further c o w e d by a positive Sakaguchi reaction. The N-terminal position of serine was confirmed by a positive Nessler test for ammonia after periodate oxidation as well as by reaction with FDNB; thus, following acid hydrolysis, DNP-serine was found.Analysis for the peptide in the TCA-soluble portion, using paper electrophoresis. indicated that the peptide occurred only during the T-8 conversion. It was not AND n _ _ 10 20 30 40 50 60 - 7 0 8 0 I- pH 5.0 +-pH 6.5-1 EFFLUENT MILLILITERS FIG. 4.-Chromatogram of peptide fraction derived from the rapid activation of chymo- trypsinogen.23 The lower chromatogram @owex-SO ion exchange resin) represents the resolution of the fractions collected from tubes 4 to 12 of the upper chromatogram (XE-64 ion exchange resin). seen in activation mixtures containing only n-chymotrypsin. Other experiments indicated its absence when a-chymotrypsin was allowed to act on chymotrypsinogen.The conclusion that the peptide is liberated during the conversion of n- chymotrypsin to the &form is further strengthened by the quantitative results shown in fig. 5, in which the rates of formation and disappearance of the electro- phoretic protein components of the activation mixtures are shown as a function of time. Also plotted in this figure are the amounts of peptide liberated during activation. In these experiments the entire activation mixture, after treatment with DFP, was applied to the XE-64 column and the peptide determined quanti- tatively by elution with 0-3 M citrate buffer. It is evident from these results that the yield of peptide follows rather closely the curve describing the formation of a-chymotrypsin. Activity measurements on a similar activation mixture indicated that the per cent of maximally attainable esterase activity corresponded approxim- ately to the amount of chymotrypsinogen disappearing.H.NEURATH A N D W. J . DREYER 39 On the basis of the data just presented it is possible to identify the segment of the peptide chain of chymotrypsinogen which is primarily affected by the rapid activation process. It will be remembered that w-chymotrypsin differs from chymotrypsinogen in possessing an N-terminal isoleucyl-valine sequence and that the conversion of 7 ~ - to the 6-form yields no new N-terminal group, a Gterminal leucine group and the dipeptide serylarginine. Furthermore, since the release of the dipeptide appears to be exclusively associated with the m-8 conversion it is unlikely that serylarginine is a C-terminal sequence in chymotrypsinogen. The present data, therefore, suggest that tryptic hydrolysis of the argin yl-isoleucine bond in the sequence leucyl-seryl-arginyl-isoleucyl-valine is the single chemical event accompanying the formation of w-chymotrypsin.The subsequent chymo- trypsin-catalyzed conversion of w to the 8 form involves the hydrolysis of the leucyl- seryl bond, giving rise to the dipeptide, serylarginine, and a Gterminal leucine group and an N-terminal isoleucyl-valine sequence in the protein. It is to be anticipated that the action of the proteolytic enzymes present in activation mixtures will be more profound the slower the rate and the longer the time of activation. This is borne out by end-group analysis which has shown that upon prolonged incubation of a rapid activation mixture S-chymotrypsin is converted to an active enzyme which contains an additional C-terminal tyrosine RAPID ACTIVATION of CHY MOTRY PS I N OG E N 0 8-CHT 0 PEPTIDE A T-CHT tf 40f Q CHTG 20 0 10 20 30 40 50 60 70 80 90 Minutes of Activation FIG. 5.-Distribution of protein and peptide components of rapid activation mixtures of chymotrypsinogen as a function of time of activation.23 RELATION TO SLOW ACTIVATION group (table 3).The number of terminal groups is further increased, to four, when slow activation mixtures are permitted to stand under conditions leading to the formation of a-chymotrypsin. No additional changes in end-groups occur during the subsequent stages of slow activation required for the isolation of /3 and y-chymotrypsin; however, this type of analysis is not conclusive since these proteins are electrophoretically heterogeneous and since by happenstance partial degradation of the proteins may have led to a moiety having qualitatively identical terminal groups.The increasing complexity of the systems resulting from prolonged activation is clearly demonstrated by electrophoresis in the moving boundary apparatus for extended periods of time.24 Representative patterns of slow activation mixtures and of crystalline chymotrypsins are shown in fig. 6. Although these patterns are more heterogeneous than those of rapid activation mixtures, there is no detectable change in the mobility of the major component; indeed, it has not been possible to resolve the major peak of a mixture of rapid and slow40 ACTIVATION OF TRYPSINOGEN activation mixtures.Electrophoretic patterns of the crystalline chymotrypsins resemble in complexity those of slow activation mixtures, All of these crystal- line proteins contain more than one electrophoretic component and certain rela- tionships among the mobiIities of the various components exist. For instance, the electrophoretic mobilities of the major components of rapid and slow activa- tion mixtures and of a-chymotrypsin are identical under the conditions of these measurements (sodium acetate buffer, pH 4.97, 0.1 ionic strength) and are some- what lower than those of the major components of B and y-chymotrypsins, which are also identical. The mobilities of the slowest moving peaks of /3- and y- chymotrypsins are identical to that of the major peak of a-chymotrypsin.It is likely that the shift to somewhat higher mobilities attending the formation of /3- and y-chymotrypsin can be related to the release of a peptide having pre- dominantly acidic properties.4 I 2 . 3 4 1 5 1 6 FIG. 6.-Electrophoretic diagrams (as- cending) of slow activation mixtures and crystalline chymotrypsins in sodium acetate buffer, pH 4-97, 0.1 ionic strength.24 (1) Rapid activation mixture, chymotrypsinogen : trypsin 33 : 1, (DIP-&chymotrypsin). (2) Slow activation mixture, chymotrypsino- gen : trypsin 5000 : 1, 28 h activation. (3) Slow activation mixture, chymo- trypsinogen: trypsin 10,OOO: 1, 87 h of activation. (4) 2 x crystallized DIP-a-chymotrypsin.(5) 2 x crystal- lized DIP-y-chymotrypsin. (6) DIP derivative of 2 x crystallized p-chy- mo trypsin . THE EFFECT OF ACTIVATION ON SEDIMENTATION BEHAVIOUR It has been shown in previous studies of a-chymotrypsin that the concentration dependence of the sedimentation constant,lS, 1 6 2 5 diffusion constant,l6 viscosity and light scattering 26 is in many ways explainable in terms of a reversible dimer- ization. The extent of dimerization was reported to decrease with increasing pH, within the range of pH 3-86 to 6.2, in contrast to solutions of chymotrypsinogen which followed approximately the course expected for a monomer. In view of the electrophoretic heterogeneity of a-chymotrypsin it was deemed of interest to re-investigate the sedimentation behaviour of the products of rapid and slow activation. The results are summarized in fig.7. In contrast to crystallhe a-chymotrypsin, the DIP-derivatives of rapid as well as slow activation mixtures (chymotrypsinogen : trypsin 5000 : 1) revealed, at pH 3-86, a sedimentation behaviour characteristic of a pure monomer. Except for DIP-/3-chymotrypsin, only one sedimenting peak was seen in all patterns of the series represented by fig. 7. However, it was of considerable interest to note that activation mixtures corresponding to DIP-, T- and 6-chymotrypsins, while monomeric at pH 3-86 revealed at pH 7.5 a concentration dependence characteristic of a monomer- dimer equilibrium. Dimerization was found to be reversible both with respect3.0- e e A 9 0 e B m 0 ra m 0 0 0 ' 2.0' pH 3.0 GLYCINE r$= 0.1 40 0 9 , - A 0 .I I I I 1 25.0 2.0-t 0 5.0 10.0 15.0 20.0 Protein Conc. mg./ mi. FIG. 7.--Concentration dependence of sedimentation constants of chymotrypsinogen, activation mixtures and crystalline chymotrypsins. The symbols refer to the following preparations : A chymotrypsinogen ; (j rapid activation mixture containing /3-phenyl- propionic acid (DIP-n-chymotrypsin) ; 0 rapid activation mixture (DIP-8-chymotrypsin) ; 0 slow activation mixture, chymotrypsinogen : trypsin, 5000 : 1, 28 h of incubation ; 0 slow activation mixture (according to Kunitz and Northrop; 2 x recrystallized DIP-cc-chymotrypsin ; <) DIP derivatives of 2 x crystallized pzhymotrypsin. activation, appears to exist only as a monomer, regardless of pH, whereas n- and 8-chymotrypsin reveal a sedimentation behaviour at pH 7.5 characteristic of a monomer-dimer equilibrium.(ii) The relatively homogeneous products of rapid activation, n- and 8-chymotrypsin, differ from the crystalline proteins in the pH dependence of dimerization. Since, in terms of end-group analysis and electro- phoresis, n- and 8-chymotrypsin are more nearly homogeneous than the crystalline enzymes, the difference in sedimentation behaviour between chymotrypsinogen and 71- and 8-chymotrypsin appears to be more pertinent to the study of the activation process. 2.5- wQ DISCUSSION Comparison of the activation of trypsinogen and chymotrypsinogen shows certain common features of unusual interest: the splitting of a singIe peptide bond suffices to convert the zymogen to the active form.This step is in both cases catalyzed by trypsin and a bond between a basic amino acid (lysine and arginine, respectively) and an isoleucyl-valine sequence is broken. It is apparent Q rI2' 0.2 - El .y42 ACTIVATION OF TRYPSINOGEN that the activating enzyme operates with a high degree of selectivity and restraint. Thus trypsinogen has sixteen peptide bonds which might conform to the specificity of trypsin (contributed by 14 lysines and 2 arginines) 13 and chymotrypsinogen has an equivalent number (contributed by 12 lysines and 4 arginines).lg Nevertheless, only one of these bonds is broken during activation. Before considering the rela- tion of this single hydrolytic event to the appearance of enzymatic activity, it might be of interest to focus attention to those features which set these two activating systems apart.In trypsinogen, the release of a peptide is an obligatory accompaniment of the activation process. It is possible that the predominantly acidic properties constitute a unique feature of this activation peptide. Thus the N-terminal portion of the polypeptide chain of trypsinogen may not fit into the helical configuration of the molecule because of the electrostatic repulsion between the four adjacent aspartic acid residues which would tend to keep this segment in a fully extended configuration. The lysyl-isoleucyl bond may thus be exposed to the action of trypsin, whereas the other 15 vulnerable bonds remain protected within the helical configuration of the molecule.When activation is carried out in the absence of calcium ions, the helical structure might become sufficiently distorted to expose these additional bonds to tryptic hydrolysis, yielding " inert protein " or more profoundly degraded products. Alternatively, it might be suggested that the N-terminal hexapeptide sequence of the trypsinogen molecule, by virtue of its acidic properties, shields electrostatically basic groups of the potentially active centre and that the removal of the peptide is the structural event responsible for activation. However, this mechanistic interpretation appears less plausible if a common fundamental mechanism is to be ascribed to the activation of the two zymogens, since the release of a peptide is not involved in the primary step of the activation of chymotrypsinogen. As already mentioned, the rapid activation of chymotrypsinogen is a stepwise process mediated by the two proteolytic enzymes trypsin and chymotrypsin ; each of these opens, in sequence, a single peptide bond.Since the two products of activation, n- and S-chymotrypsin, have identical specific enzymatic activities the molecular changes responsible for the appearance of catalytic activity must be associated with the trypsin-catalyzed hydrolysis of the arginyl-isoleucine bond. Several related interpretations may be considered. From a chemical viewpoint it may be significant that trypsin and all active forms of chymotrypsin possess the N-terminal isoleucyl-valine sequence. If this configuration were part of the " active centre ", removal or chemical modification of these amino acid residues should have a profound effect on enzymatic activity. Alternatively it may be suggested that cleavage of the strategically located peptide bond may unmask a preexisting structural region responsible for catalytic activity; or else, more or iess profound intramolecular changes attending the cleavage of the arginyl-iso- leucine bond may create a configuration endowed with enzymatic functions.Such structural differences as may exist between the zymogen and the active enzyme might be revealed by X-ray diffraction analysis, and are perhaps also reflected in the tendency of the T- and 8-chymotrypsins, in contrast to chymotrypsinogen, to undergo reversible dimerization at pH 7.5.* It is worthy of note that active trypsin, unlike trypsinogen, is also subject to molecular association.*2 * Recent experiments in this laboratory indicate that the activation of both chymo- trypsinogen and trypsinogen is associated with a sigmficant decrease in optical laevo- rotation.29 In terms of the work of Kauzmann and co-workers27 these observations may be interpreted as indicative of structural rearrangements of the protein molecules.Thus the zymogens could be considered to be held in somewhat constrained configurations which are free to rearrange toward more highly folded configurations when the appropriate peptide bonds are broken, However, the greater sensitivity of the optical rotation of the active enzyme (S-chymotrypsin) as compared to the zymogen (chymotrypsinogen), to change in pH might hply, at the same time, a greater flexibility in the structure of the enzyme.H .NEURATH AND W. J . DREYER 43 As previously mentioned, trypsinogen can be activated also by enterokinase and by a pencillium kinase and, as recently reported, chymotrypsinogen can also be activated by a protease from B. subtiZis.28 It is to be expected that investigation of these activation processes by methods similar to those reported herein, will aid in the evaluation of the present conclusions and expand on their significance. The unpublished work described in this paper has been supported in part by the United States Public Health Service, research grant C-2286 and by funds made available by the people of the State of Washington, Initiative 171. 1 Northrop, Kunitz and Herriott, Crystalline Enzymes (Columbia University Press, 2 Green and Neurath, in The Proteins, ed.Neurath and Bailey (Academic Press, 3 Balls and Jansen, Adv. in Enzymol., 1952, 13, 321. 4 Neurath, Gladner and Davie, in The Mechanism of Enzyme Action, ed. McElroy 5 Desnuelle and Rovery, in The Chemical Structure of Proteins, ed. Wolstenholme 6 Bettelheim and Neurath, J. Biol. Chem., 1955, 212, 241. 7 Davie and Weurath, J. Biol. Chem., 1955, 212, 515. 8 Sanger, Biochem. J., 1945, 39, 507 ; 1949, 45, 126, 563. 9 Gladner and Neurath, J. Biol. Chem., 1953,205, 345 ; 1954,206,911. loTietze, J. Biol. Chem., 1953, 204, 1. 11 Cunningham, J. Biol. Chem., 1954, 211, 13. 12 Cunningham, Tietze, Green and Neurath, Faraday Soc. Discussions, 1953, 13, 58. 13 Cohen and Neurath, unpublished experiments. 14 Jacobsen, Compt. rend. trm. Lab. Carlsberg, Serie Chim., 1947, 25, 325. 15 Schwert, J. Biol. Chem., 1949, 179, 655. 16 Schwert and Kaufman, J . Biol. Chem., 1951, 190, 799, 807. 17 Rovery, Fabre and Desnuelle, Biochim. Biophys. Acta, 1953, 10,481 ; 12, 547. 18 Hirs, J. Biol. Chem., 1953, 205, 93. 19 Wilcox and Cohen, unpublished experiments ; see also ref. (2), table 3. 20 Bettelheim, J. Biol. Chem., 1955, 212, 235. 21 Rovery and Desnuelle, Biochim. Biophys. Acta, 1954, 13, 300. 22 Rovery, Poilroux and Desnuelle, Biochim. 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