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Observations on the electrophoretic and ultracentrifugal changes accompanying the activation of chymotrypsinogen
ARCHIVES
OF
BIOCEEMISTRY
AND
BIOPHYSICS
69, 146-166 (1966)
Observations on the Electrophoretic and Ultracentrifugal Changes Accompanying the Activation of Chymotrypsinogenl William J. Dreyer, Roger D. Wade and Hans Neurath From the Department of Biochemistry, University of Washington, Seattle, Washington Received
June 6, 1966
Recent investigations have revealed striking differences between chymotrypsinogen and the crystalline product of slow activation, cr-chymotrypsin, with respect to both ultracentrifugal (l-3) and electrophoretic behavior (4, 5). Thus it has been shown in studies of a-chymotrypsin that the concentration dependence of the sedimentation constant, diffusion constant, viscosity (2), and light scattering (6) was in many ways explainable in terms of a reversible dimerization. This was particularly pronounced at pH 3.86, in contrast to solutions of chymotrypsinogen, which followed approximately the course expected for a monomer. It has also been recently reported that cY-chymotrypsin presents a complex electrophoretic pattern which includes, besides a major component, several other components, all of these being enzymatically active (4). In view of the widespread interest in the physical and chemical changes attending the activation of chymotrypsinogen, we should like to report in the present communication the electrophoretic and ultracentrifugal behavior of solutions of activation mixtures. These studies, carried out as part of a more general study of the conversion of precursors of proteolytic enzymes to the active forms (7-9), have shown that various activation mixtures are more homogeneous than the crystalline enzymes and differ from them in sedimentation behavior. 1 This work 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. 146
146
DREYER,
WADE
AND NETJFUTH
EXPERIMENTAL
Methods Sedimentation analyses were carried out in the Spinco, model E, ultracentrifuge at 59,780 r.p.m. For solutions of low pH a sector-shaped cell, having a plastic Kel-F center section, 12 mm. thick, was employed. In a few cases the synthetic boundary cell was used. Sedimentation constants were calculated from leastsquare slopes, using the equation: log x = s3t + c where x is the radial distance from the peak to the center of rotation at time t, w the angular velocity, and s the sedimentation constant. Sedimentation constants were corrected to 20” and to water as a solvent, using Eq. (173) of Svedberg and Pedersen (10). Unless otherwise noted, sedimentation runs were restricted to a period of approximately 1 hr. in order to limit the temperature change of the rotor to not more than 0.5’. Viscosity measurements were carried out in Ostwald viscometers at 20 f 0.005”. Density measurements were carried out in pycnometers of 10 ml. capacity at 2o”. Electrophoresis. Electrophoretic measurements were carried out at lo in the Spinco, model H, electrophoresis apparatus. For adequate resolution of the components, electrophoresis was extended to a period of time up to 24 hr. using electrolytic compensation to maintain the boundaries within view at all times. The reported electrophoretic mobilities were calculated in the conventional manner from ascending boundary patterns before counter compensation was started, since these showed better resolution of the components.
Materials Chymotrypsinogen was prepared by seven recrystallizations with ammonium sulfate (ll), followed by two recrystallizations from ethanol (12). Crystalline Q-, fl-, and r-chymotrypsins were prepared* by published methods (11). DIP*-aand y-chymotrypsins were prepared by the addition of DFP at O’, to solutions of the respective crystalline active enzymes at pH 7.6-7.8. The DIP-enzymes were then twice recrystallized by methods similar to those used for the preparation of the active enzymes and, after dialysis against 0.001 N HCl, they were lyophilized. DIP-fl-chymotrypsin was simply prepared by the addition of an excess of DFP to solutions of the twice-crystallized enzyme at O”, pH 7.6-7.8. After lowering the pH to 3.0 with 1 N HCl, the solutions were dialyzed against 0.001 N HCI and then used as such. Actiuation. The composition of activation mixtures was as follows: Chymo* We are indebted to Dr. J. A. Gladner for the preparation of crystalline @and r-chymotrypsins. * The following abbreviations are used: DFP, diisopropyl phosphofluoridate; DIP, diisopropylphosphoryl.
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trypsinogen, 40 mg./ml., sodium phosphate btier, pH 7.8,0.05 M; activation was carried out at 0”. For rapid activation (7,13), the chymotrypsinogen-trypsin mole ratio was 33:1, whereas for slow activation this ratio was increased to 5OOO:l and 1OOOO:l.In one specifically stated instance, the composition of the slow activation mixture was as given by Northrop, Kunits, and Herriott (11). When x-chymotrypsin was the desired end product, 0.1 or 0.2 M sodium &phenylpropionate was also added to the rapid activation mixtures (7,9). Activation was stopped by the addition of a 15- or 50-fold molar excess of a 1 M solution of DFP in isopropyl alcohol. After 30 min. standing at 0”, the pH was adjusted to that of the buffer
to be used for subsequent physical measurements, and the solutions were equilibrated by dialysis in the cold against the corresponding buffer.
Protein concentrations were determined spectrophotometrically, using an extinction coefficient of E:E ,,,#= 20.6for chymotrypsinogen (2) and chymotrypsins, and E:To mP= 14.4 for trypsin (8). RESULTS
Electrophoresis In a recent publication, the changes in electrophoretic pattern which accompany the rapid activation of chymotrypsinogen under various conditions have been described (9). For purposes of comparison with slow activation and the crystalline enzymes, representative patterns of chymotrypsinogen, ?r- and d-chymotrypsin are included in Fig. 1. It will be noted that this preparation of chymotrypsinogen is electrophoretically nearly homogeneous (at pH 4.97). Although it was previously reported that under these conditions chymotrypsinogen and n-chymotrypsin have identical mobilities (7), upon prolonged electrophoresis full separation of these two components occurs. In the experiment represented by Fig. 1, diagram 2, 0.1 M /3-phenylpropionate was present in the activation mixture and the reaction was stopped by the addition of DFP before conversion of chymotrypsinogen to a-chymotrypsin was complete. It will be noted that under these conditions the component corresponding to 6chymotrypsin is just barely visible (see also Table I). The electrophoretic pattern corresponding to DIP-Schymotrypsin (diagram 3) reveals a major component comprising approximately 90% of the total protein. The major peak remains symmetrical even after electrophoresis for 20 hr. Diagrams 4 and 5 of Fig. 1 show the electrophoretic pattern of slow activation mixtures. It is apparent that as the rate of activation decreases, and the time increases, the electrophoretic patterns of the activation mixtures become more heterogeneous (compare diagrams 3, 4, and 5 of Fig. 1). There is no detectable change in the mobility of the major component and, indeed, it has not been possible to resolve the major peak of
148
DREYER,
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NEURATH
FIQ. 1. Ascending electrophoretic patterns of chymotrypsinogen and activation mixtures in a sodium acetate buffer, pH 4.97,0.1 ionic strength. Direction of migration from right to left, at 5.7 v. cm.-‘. Diagrams l-5 represent the following
conditions: (I) chymotrypsinogen; (8) a rapid activation mixture, containing in decreasing order of mobility, chymotrypsinogen, DIP-r-chymotrypsin, and a trace of DIP-i-chymotrypsin; (S) a rapid activation mixture containing DIPb-chymotrypsin; (4) a slow activation mixture (chymotrypsinogen-trypsin 5000:1,
23 hr. of incubation); (6) a slow activation mixture (chymotrypsinogen-trypsin lO,OOO:l, 87 hr. of incubation). In diagrams 1,3,4,, and 5 the 6 boundary appears at the extreme right. The times of electrophoresis were 445,1357,596,449,and 456 min., respectively, in diagrams l-5.
a mixture of rapid and slow activation products (e.g., corresponding to those illustrated in diagrams 3 and 4). It is worthy of note that the peak migrating just ahead of the major peak of slow activation mixtures corresponds in mobility to the major peak seen in electrophoretic patterns of crystalline /3- and y-chymotrypsins (see below). If a slow activation mixture, prepared according to Kunitz and Northrop (1 l), was examined eiectrophoretically after 2 and 17 days of incubation, respectively, progressive conversion of the peak corresponding in mobility to a-chymotrypsin to that of p- and y-chymotrypsin could be observed. Representative electrophoretic patterns of the crystalline chymotrypsins are shown in Fig. 2. All of these crystalline proteins contain more than one electrophoretic component. It is of particular interest that CYchymotrypsin which has been investigated by such a variety of methods is, like its DIP derivative, electrophoretically heterodisperse. The mo-
ACTIVATION
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149
CHYMOTRYPSINOGEN
TABLE I Electrophoretic Protein
Mobilitiesa
of
= I
Chymotrypsinogen
preparation
.Chymotrypsinogen Rapid activation mixture with &phenyl. propionic acid +chymotrypsin)
and Chymotrypsins
Mobility
(ascending)
-7
3.8
3.65
3.5
3.2s
Other
Mb M
t
Rapid activation mixture (a-chymotryp. sin)
M
Slow activation mixture (28 hr. incubachymotrypsinogen : trypsin tion, 5000 2) Slow activation mixture (87 hr. incubatrypsin chymotrypsinogen: tion, 10,000:1) DIP-or-chymotrypsin, 2 X crystallized DIP-fl-chymotrypsin DIP-r-chymotrypsin, 2 X crystallized
M m
M
t
M t m
m’
E M
= Values given in 10T6 cm.z V.-l sec.-I, in a sodium acetate buffer, pH 4.97,0.1 ionic strength. * M = major component; m and m’ = minor components; t = trace compon-
ent. See also the diagrams in Figs. 1 and 2. bility of the fastest moving component corresponds to that of the major component of /3- and y-chymotrypsins, while that of the major component corresponds to that of the major component of both rapid and slow activation mixtures. The electrophoretic mobilities of the major component of each p- and y-chymotrypsin are identical and the patterns reveal certain similarities. In each case the mobility of the slowest moving peak is identical to that of the major peak of ol-chymotrypsin. Table I summarizes the relations of the electrophoretic mobilities of the components in the various preparations which have been investigated. Since the mobilities of several of these components are so close to each other and since the observed mobilities are somewhat dependent on protein concentration and relative component distribution (14), it was not possible to characterize a component by mobility alone. In addition, it was necessary to rely on the comparison of patterns of two protein solutions with each other and with that of a mixture containing a known fraction of each. The similar electrophoretic behavior of DIP-p- and-ychymotrypsin is also evidenced by the electrophoretic pattern shown in
150
DREYER,
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AND NEURATH
FIG. 2. Ascending electrophoretic patterns of DIP derivatives of crystalline chymotrypsins, in a sodium acetate buffer, pH 4.97, 0.1 ionic strength. Direction of migration from right to left, at 5.7 v. cm.+. Diagrams 14 represent the following conditions: (1) 2X crystallized DIP-cY-chymotrypsin; (9) DIP derivative of 2X crystallized &chymotrypsin; (5) 2~ crystallized DIP-r-chymotrypsin; (4) mixture of B part of DIP-B- and g of DIP-r-chymotrypsin. The times of electrophoresis were 1485, 1480, 1405, and 1350 min., respectively, for diagrams 14. The 6 boundary is not visible in the patterns due to countercurrent electrophoresis. The pattern at the bottom of diagram 4 is a representative Rayleigh fringe pattern.
Fig. 2, diagram 4, which has been obtained from a mixture containing N part of /3- and $5 part of y-DIP-chymotrypsin. Even upon prolonged electrophoresis (1350 min.) no resolution of the major peak could be observed. This finding is of particular interest since these two proteins have been found to have identical N- and C-terminal groups (15, 16). Sedimentation Figure 3 shows the concentration dependence of sedimentation constants of activation mixtures and crystalline proteins in buffered solutions of different pH. DIP-cY-chymotrypsin, at pH 3.86 (0.02 M sodium acetate, 0.18 M NaCl), shows the relatively high sedimentation constant which has been previously attributed to dimerization. However, the concentration dependence, though similar to that previously reported from this laboratory (16), is at variance with that described earlier by Schwert (l), since it shows a positive rather than a negative slope in the concentration range above 5 mg./ml. Similar sedimentation behavior was found for cY-chymotrypsin though variation among different preparations was observed. In contrast to these crystalline proteins, the DIP derivatives
ACTIVATION
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151
CHYMOTRYPSINOGEN pH 3.66 ACETATE
3.5
1
25J 3.5
l/
3.0
**
-
rt2.a2
*
Q 0 = ” 0 0
*
a
@
-
pH 7.5 PHOSPHATE w2. 0.1
PH 3.66
ACETATE
rf2’ 0.2
*
2.5-v
-
2.5 2.0
0
5.0
10.0 15.0 Protein Cont. mg./ ml.
20.0
25.0
dependence of sedimentation constants of chymotrypFIG. 3. Concentration sinogen, activation mixtures and crystalline chymotrypsins. The symbols refer to the following preparations: A chymotrypsinogen; @ rapid activation mixture containing /3-phenylpropionic acid (DIP-r-chymotrypsin) ; 0 rapid activation mixture (DIP-&chymotrypsin) ; q slow activation mixture, chymotrypsinogentrypsin, 5000:1, 28 hr. of incubation; q slow activation mixture [according to Kunita and Northrop (ll)]; 0 2X recrystallized DIP-a-chymotrypsin; q DIP derivative of 2 X crystallized ,%chymotrypsin.
of rapid as well as slow activation tation
constants
characteristic
mixtures4 revealed at this pH sedimen-
of that of a pure monomer (Fig. 3). How-
ever, a slow activation mixture prepared according to Kunitz and Norshowed a concentration dependence of throp [quoted in Ref. (ll)] sedimentation constants intermediate between that of chymotrypsinogen and DIP-a-chymotrypsin. After extrapolation to zero protein concentration, the sedimentation constant of DIP-&chymotrypsin wax Szo,W= 2.60. Only one sedimenting peak was seen in all patterns of the series represented by Fig. 3. It has been previously reported that the degree of dimerization of cr-chymotrypsin decreases with increasing pH within the pH range of 3.8-7.8 (1, 6). It was, therefore, of considerable interest to note that an activation mixture corresponding to DIP-T- and -6-chymotrypsin, respectively, while monomeric at pH 3.86, at pH 7.5 (K2HP04, 0.0308 M; KH~PO4,0.0072 M) revealed a concentration dependence characteristic 4 (chymotrypsinogen:trypsin,
5OOO:l)
152
DREYER,
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NEURATII
Fm. 4. Representative sedimentation patterns of DIP-chymotrypsins. (1) and (4) are rapid activation mixtures (DIP-khymotrypsins), at pH 3.86, 74 and 154 min., respectively, after reaching full speed; (2) and (6), the same preparation at pH 7.5, 74 and 146 min., respectively, after reaching full speed; (7) the same preparation at pH 3.0,70 min. after reaching full speed; (8) the same preparation in a synthetic b,oundary cell at pH 7.5,36 min. after reaching full speed. Protein concentration above synthetic boundary 12 mg./ml., and below the boundary, 16 mg./ml. (3) and (6) 2~ crystallized DIP-a-chymotrypsin, at pH 3.86,62 and 160 min. after reaching full speed. Protein concentration in runs 1,2,3 and 7 was 19,21, 17, and 20 mg./ml., respectively. The direction of sedimentation was from right to left.
of a monomer-dimer equilibrium (Fig. 3). These results were strictly reproducible; the observed sedimentation constants were independent of the time elapsed after preparation of the solutions (148 hr.). Furthermore, when a solution of DIP-&chymotrypsin, prepared at a high concentration, was diluted to a lower range, the sedimentation constant decreased in accordance with the plotted relation, demonstrating the ready reversibility of dimerization upon dilution. The pa-dependence of sedimentation constants of DIP-&chymotrypsin was also found to be strictly reversible. When a solution at pH 7.5 was equilibrated by dialysis against a pH 3.86 buffer and examined again in the ultracentrifuge, the results were identical with those obtained for solutions which were initially adjusted to pH 3.86. At pH 3.0 (0.02 M HCl, 0.08 M NaCl, titrated to pH
ACTIVATION OF CHYMOTRYP~INOGEN
153
3 with glycine) chymotrypsinogen, DIP derivatives of rapid and slow activation mixtures and crystalline DIP-W and -@-chymotrypsin are indistinguishable from one another in sedimentation behavior. The concentration dependence curve (Fig. 3) reveals the existence of a monomer throughout the entire concentration range studied. Thus a more reliable comparison of the sedimentation constants of chymotrypsinogen and the various chymotrypsins can be made, yielding a linearly extrapolated sedimentation constant of ~920,~= 2.46 S. This lower value, as compared to those found at the higher pH’s, may be due to the high charge on the protein. Representative sedimentation patterns are shown in Fig. 4. It will be noted that when the sedimentation constant shows a concentrat,ion dependence characteristic of a monomer-dimer equilibrium, the sedimenting boundary is distinctly asymmetric, with a trailing edge toward the solvent side. This is particularly evident in runs extended for longer periods of time (Fig. 4, patterns 5 and 6), but is also apparent when shorter term runs of DIP-&chymotrypsin at pH 7.5 (patterns 2 and 8) or DIP-a-chymotrypsin at 3.86 (pattern 3) are compared with DIP-& chymotrypsin at pH 3.86 or pH 3.0 (patterns 1,4, and 7). DIP-cu-chymotrypsin, which is also monomeric at pH 3.0, gave also a symmetrical boundary pattern at that pH. Included in Fig. 4 is a pattern obtained on DIP-d-chymotrypsin, pH 7.5, with the use of a synthetic boundary cell (17) (pattern 8). Because of the short duration of the run, the asymmetry to be expected for the boundary between protein and buffer is just barely noticeable, whereas, in accordance with expectation, no asymmetry is discernible in the synthetic boundary where protein concentration is relatively high on both sides of the boundary and is in a region where the slope of the concentration dependence curve is relatively small.
DISCUSSION The electrophoretic heterogeneity of crystalline a-chymotrypsin renders the interpretation of previous physical studies of this protein of doubtful significance. In contrast, the DIP derivatives of the major enzymatic components of rapid activation mixtures, i.e., rr- and d-chymotrypsins, were relatively homogeneous in electrophoresis. However, the heterogeneity increased as the time of activation increased and its rate decreased, probably due to secondary proteolytic degradation of the enzymes. It is of interest to note that the more heterogeneous activation mixtures do not readily lend themselves to fractionation by repeated crystallization.
154
DREYER,
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NEURATH
As evidenced by the data given in Table I, activation is accompanied by changes in electrophoretic mobility. The largest change, occurring during the conversion of r-chymotrypsin to the &form, can now be clearly ascribed to the liberation of the basic dipeptide, serylarginine (9). The mobility of the major protein component remains constant through the succeeding stages of activation leading to the formation of a-chymotrypsin. The subsequent shift to somewhat higher mobilities attending the formation of @-and y-chymotrypsin may be related to the release of a peptide having predominantly acidic properties. There is disconcerting disagreement among various investigators concerning the sedimentation behavior of crystalline cu-chymotrypsin (l-3, 6, 16). The most that can be said is that the exact shape of the curve relating sedimentation constants to protein concentration, though not clearly defined, varies with pH, the highest sedimentation constant being observed at about pH 3.86. In contrast, chymotrypsinogen, d-chymotrypsin, and relatively slow activation mixtures (28 hr. of activation, chymotrypsinogen-trypsin mole ratio 5000: 1) all reveal at pH 3.86 a sedimentation pattern characteristic of a monomer. The common feature of these proteins is that they are relatively homogeneous in electrophoresis and contain little, if any, @-or y-chymotrypsin. The slower, and more heterogeneous activation mixtures, prepared according to Kunitz and Northrop (1 l), contain more /3- or y-chymotrypsins and their sedimentation behavior resembles more nearly that of the crystalline (Y- and /3forms (Fig. 1). Thus the relative component distribution of a preparation appears to have a profound influence on its sedimentation behavior. In more alkaline solutions, i.e., pH 7.5, dimerization of cr-chymotrypsin decreases, whereas that of ?r- and Schymotrypsin increases. Chymotrypsinogen, however, remains as a monomer regardless of pH. It is, therefore, probable that the appearance of new terminal groups during activation is functionally related to the ability of the protein to dimerize. Since each of the enzymes appears to have its own characteristic pH dependence of dimerization, no generalization concerning the groups responsible for this process can yet be made. s However, since in terms of end-group analysis (7) and electrophoresis, ?r- and &chymotrypsins are 6 Although, after removal of the two C-terminal groups of DIP-a-chymotrypsin by carboxypeptidase the protein no longer dimerizea at pH 3.86 (16), the sedimentation constant increases with increasing protein concentration at pH 5.0, and more so at pH 6.2 (unpublished observations by Dr. J. Kraut). Since no electrophoretic patterns of these preparations are available, further interpretation of these findings is not warranted.
ACTIVATION
OF CHYMOTRYPSINOGEN
155
more nearly homogeneous than the crystalline (Y- and ,&forms (15, IS), the difference in sedimentation behavior between chymotrypsinogen and ?r-chymotrypsin appears to be more significant for the study of the activation process than is the difference between 6- and cY-chymotrypsins. In the light of recent work on the nature of the chemical changes accompanying the activation of chymotrypsinogen (7, 9), it is clear that the molecular weights of chymotrypsinogen and of r- and I-chymotrypsins are nearly identical, chymotrypsinogen differing from ?r- and 6chymotrypsin by 18 and 279, respectively. Hence, in view of the virtual identity of the extrapolated sedimentation constants at pH 3, the change in frictional ratios accompanying activation must be likewise negligibly small. Furthermore, since the extrapolated sedimentation constants of (Y- and @-chymotrypsins are indistinguishable from those of the above proteins, it seems probable that the proteolytic degradation attending the formation of the crystalline enzymes does not result in a large change in molecular weight. SUMMARY
Electrophoretic and ultracentrifugal measurements of chymotrypsinogen, of rapid and slow activation mixtures, and of crystalline chymotrypsins are described. Whereas chymotrypsinogen and rapid activation mixtures show a relatively high degree of electrophoretic homogeneity, the electrophoretic patterns become more complex and heterogeneous as the time of activation increases and the rate decreases. The DIP derivatives of crystalline, (Y-, fl-, and y-chymotrypsins are among the most heterogeneous products of this series of proteins. All of these proteins exist as a monomer at pH 3, within the concentration range of 2-20 mg./ml. In contrast to chymotrypsinogen, which is monomeric over the entire pH range studied, DIP-r- and -b-chymotrypsins at pH 7.5 exist in concentration-dependent monomer-dimer equilibrium. The pH dependence of dimerization of the crystalline enzymes differs markedly from those of the above proteins. REFERENCES 1. SCAVERT, G. W., J. Biol. Chem. 179,655 (1949). 2. S~HWERT, G. W., AND KAUFMAN, S., J. Biol. Chem. 190,799,807 (1951). 3. SMITH, E. L., AND BROWN, D. M., J. Biol. Chem. 196, 525 (1952). 4. EOAN, R., Federation Proc. 12, 199 (1953); 14, 206 (1955). 5. EGAN, R., MICHEL, H. O., AND SCXLUETER, R., Federation Proc. 13,202 (1954). 6. STEINER, R., Arch. Biochem. and Biophys. 63, 457 (1954).
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NEURATH
7. BETTELHEIM, F. R., AND NEURATH, H., J. Biol. Chem. 212,241 (1955). 8. DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 515 (1955). 9. DREYER, W. J., AND NEURATH, H., J. Am. Chem. Sot. 77, 814 (1955); J. Biol. Chem. in press. 10. SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge.” Oxford University Press, Oxford, 1940. 11. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., “Crystalline Enzymes.” Columbia University Press, New York, 1948. 12. KUNITZ, M., J. Gen. Physiol. 32, 265 (1948). 13. JACOBSEN, C. F., Compt. rend. trau. lab. Carlsberg, SCr. chim. 26, 325 (1947). (N eurath, H. and Bailey, K., eds.), Vol. 14. ALBERTY, R. A., in “The Proteins,” I, Chap. 6. Academic Press, New York, 1953. 15. ROVERY, C., FABRE, C., AND DESNUELLE, P., Biochim. et Biophys. Acta 10, 481 (1953). 16. GLADNER, J. A., AND NEURATH, H., J. Biol. Chem. 206,911 (1954). 17. PICKELS, E. G., HARRIN~TON, W. F., AND SCHACHMAN, H. K., Proc. Natl. Acad. Sci. U. S. 36,943 (1952).
OF
BIOCEEMISTRY
AND
BIOPHYSICS
69, 146-166 (1966)
Observations on the Electrophoretic and Ultracentrifugal Changes Accompanying the Activation of Chymotrypsinogenl William J. Dreyer, Roger D. Wade and Hans Neurath From the Department of Biochemistry, University of Washington, Seattle, Washington Received
June 6, 1966
Recent investigations have revealed striking differences between chymotrypsinogen and the crystalline product of slow activation, cr-chymotrypsin, with respect to both ultracentrifugal (l-3) and electrophoretic behavior (4, 5). Thus it has been shown in studies of a-chymotrypsin that the concentration dependence of the sedimentation constant, diffusion constant, viscosity (2), and light scattering (6) was in many ways explainable in terms of a reversible dimerization. This was particularly pronounced at pH 3.86, in contrast to solutions of chymotrypsinogen, which followed approximately the course expected for a monomer. It has also been recently reported that cY-chymotrypsin presents a complex electrophoretic pattern which includes, besides a major component, several other components, all of these being enzymatically active (4). In view of the widespread interest in the physical and chemical changes attending the activation of chymotrypsinogen, we should like to report in the present communication the electrophoretic and ultracentrifugal behavior of solutions of activation mixtures. These studies, carried out as part of a more general study of the conversion of precursors of proteolytic enzymes to the active forms (7-9), have shown that various activation mixtures are more homogeneous than the crystalline enzymes and differ from them in sedimentation behavior. 1 This work 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. 146
146
DREYER,
WADE
AND NETJFUTH
EXPERIMENTAL
Methods Sedimentation analyses were carried out in the Spinco, model E, ultracentrifuge at 59,780 r.p.m. For solutions of low pH a sector-shaped cell, having a plastic Kel-F center section, 12 mm. thick, was employed. In a few cases the synthetic boundary cell was used. Sedimentation constants were calculated from leastsquare slopes, using the equation: log x = s3t + c where x is the radial distance from the peak to the center of rotation at time t, w the angular velocity, and s the sedimentation constant. Sedimentation constants were corrected to 20” and to water as a solvent, using Eq. (173) of Svedberg and Pedersen (10). Unless otherwise noted, sedimentation runs were restricted to a period of approximately 1 hr. in order to limit the temperature change of the rotor to not more than 0.5’. Viscosity measurements were carried out in Ostwald viscometers at 20 f 0.005”. Density measurements were carried out in pycnometers of 10 ml. capacity at 2o”. Electrophoresis. Electrophoretic measurements were carried out at lo in the Spinco, model H, electrophoresis apparatus. For adequate resolution of the components, electrophoresis was extended to a period of time up to 24 hr. using electrolytic compensation to maintain the boundaries within view at all times. The reported electrophoretic mobilities were calculated in the conventional manner from ascending boundary patterns before counter compensation was started, since these showed better resolution of the components.
Materials Chymotrypsinogen was prepared by seven recrystallizations with ammonium sulfate (ll), followed by two recrystallizations from ethanol (12). Crystalline Q-, fl-, and r-chymotrypsins were prepared* by published methods (11). DIP*-aand y-chymotrypsins were prepared by the addition of DFP at O’, to solutions of the respective crystalline active enzymes at pH 7.6-7.8. The DIP-enzymes were then twice recrystallized by methods similar to those used for the preparation of the active enzymes and, after dialysis against 0.001 N HCl, they were lyophilized. DIP-fl-chymotrypsin was simply prepared by the addition of an excess of DFP to solutions of the twice-crystallized enzyme at O”, pH 7.6-7.8. After lowering the pH to 3.0 with 1 N HCl, the solutions were dialyzed against 0.001 N HCI and then used as such. Actiuation. The composition of activation mixtures was as follows: Chymo* We are indebted to Dr. J. A. Gladner for the preparation of crystalline @and r-chymotrypsins. * The following abbreviations are used: DFP, diisopropyl phosphofluoridate; DIP, diisopropylphosphoryl.
ACTIVATION OF CHYMOTRYPSINOGEN
147
trypsinogen, 40 mg./ml., sodium phosphate btier, pH 7.8,0.05 M; activation was carried out at 0”. For rapid activation (7,13), the chymotrypsinogen-trypsin mole ratio was 33:1, whereas for slow activation this ratio was increased to 5OOO:l and 1OOOO:l.In one specifically stated instance, the composition of the slow activation mixture was as given by Northrop, Kunits, and Herriott (11). When x-chymotrypsin was the desired end product, 0.1 or 0.2 M sodium &phenylpropionate was also added to the rapid activation mixtures (7,9). Activation was stopped by the addition of a 15- or 50-fold molar excess of a 1 M solution of DFP in isopropyl alcohol. After 30 min. standing at 0”, the pH was adjusted to that of the buffer
to be used for subsequent physical measurements, and the solutions were equilibrated by dialysis in the cold against the corresponding buffer.
Protein concentrations were determined spectrophotometrically, using an extinction coefficient of E:E ,,,#= 20.6for chymotrypsinogen (2) and chymotrypsins, and E:To mP= 14.4 for trypsin (8). RESULTS
Electrophoresis In a recent publication, the changes in electrophoretic pattern which accompany the rapid activation of chymotrypsinogen under various conditions have been described (9). For purposes of comparison with slow activation and the crystalline enzymes, representative patterns of chymotrypsinogen, ?r- and d-chymotrypsin are included in Fig. 1. It will be noted that this preparation of chymotrypsinogen is electrophoretically nearly homogeneous (at pH 4.97). Although it was previously reported that under these conditions chymotrypsinogen and n-chymotrypsin have identical mobilities (7), upon prolonged electrophoresis full separation of these two components occurs. In the experiment represented by Fig. 1, diagram 2, 0.1 M /3-phenylpropionate was present in the activation mixture and the reaction was stopped by the addition of DFP before conversion of chymotrypsinogen to a-chymotrypsin was complete. It will be noted that under these conditions the component corresponding to 6chymotrypsin is just barely visible (see also Table I). The electrophoretic pattern corresponding to DIP-Schymotrypsin (diagram 3) reveals a major component comprising approximately 90% of the total protein. The major peak remains symmetrical even after electrophoresis for 20 hr. Diagrams 4 and 5 of Fig. 1 show the electrophoretic pattern of slow activation mixtures. It is apparent that as the rate of activation decreases, and the time increases, the electrophoretic patterns of the activation mixtures become more heterogeneous (compare diagrams 3, 4, and 5 of Fig. 1). There is no detectable change in the mobility of the major component and, indeed, it has not been possible to resolve the major peak of
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FIQ. 1. Ascending electrophoretic patterns of chymotrypsinogen and activation mixtures in a sodium acetate buffer, pH 4.97,0.1 ionic strength. Direction of migration from right to left, at 5.7 v. cm.-‘. Diagrams l-5 represent the following
conditions: (I) chymotrypsinogen; (8) a rapid activation mixture, containing in decreasing order of mobility, chymotrypsinogen, DIP-r-chymotrypsin, and a trace of DIP-i-chymotrypsin; (S) a rapid activation mixture containing DIPb-chymotrypsin; (4) a slow activation mixture (chymotrypsinogen-trypsin 5000:1,
23 hr. of incubation); (6) a slow activation mixture (chymotrypsinogen-trypsin lO,OOO:l, 87 hr. of incubation). In diagrams 1,3,4,, and 5 the 6 boundary appears at the extreme right. The times of electrophoresis were 445,1357,596,449,and 456 min., respectively, in diagrams l-5.
a mixture of rapid and slow activation products (e.g., corresponding to those illustrated in diagrams 3 and 4). It is worthy of note that the peak migrating just ahead of the major peak of slow activation mixtures corresponds in mobility to the major peak seen in electrophoretic patterns of crystalline /3- and y-chymotrypsins (see below). If a slow activation mixture, prepared according to Kunitz and Northrop (1 l), was examined eiectrophoretically after 2 and 17 days of incubation, respectively, progressive conversion of the peak corresponding in mobility to a-chymotrypsin to that of p- and y-chymotrypsin could be observed. Representative electrophoretic patterns of the crystalline chymotrypsins are shown in Fig. 2. All of these crystalline proteins contain more than one electrophoretic component. It is of particular interest that CYchymotrypsin which has been investigated by such a variety of methods is, like its DIP derivative, electrophoretically heterodisperse. The mo-
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CHYMOTRYPSINOGEN
TABLE I Electrophoretic Protein
Mobilitiesa
of
= I
Chymotrypsinogen
preparation
.Chymotrypsinogen Rapid activation mixture with &phenyl. propionic acid +chymotrypsin)
and Chymotrypsins
Mobility
(ascending)
-7
3.8
3.65
3.5
3.2s
Other
Mb M
t
Rapid activation mixture (a-chymotryp. sin)
M
Slow activation mixture (28 hr. incubachymotrypsinogen : trypsin tion, 5000 2) Slow activation mixture (87 hr. incubatrypsin chymotrypsinogen: tion, 10,000:1) DIP-or-chymotrypsin, 2 X crystallized DIP-fl-chymotrypsin DIP-r-chymotrypsin, 2 X crystallized
M m
M
t
M t m
m’
E M
= Values given in 10T6 cm.z V.-l sec.-I, in a sodium acetate buffer, pH 4.97,0.1 ionic strength. * M = major component; m and m’ = minor components; t = trace compon-
ent. See also the diagrams in Figs. 1 and 2. bility of the fastest moving component corresponds to that of the major component of /3- and y-chymotrypsins, while that of the major component corresponds to that of the major component of both rapid and slow activation mixtures. The electrophoretic mobilities of the major component of each p- and y-chymotrypsin are identical and the patterns reveal certain similarities. In each case the mobility of the slowest moving peak is identical to that of the major peak of ol-chymotrypsin. Table I summarizes the relations of the electrophoretic mobilities of the components in the various preparations which have been investigated. Since the mobilities of several of these components are so close to each other and since the observed mobilities are somewhat dependent on protein concentration and relative component distribution (14), it was not possible to characterize a component by mobility alone. In addition, it was necessary to rely on the comparison of patterns of two protein solutions with each other and with that of a mixture containing a known fraction of each. The similar electrophoretic behavior of DIP-p- and-ychymotrypsin is also evidenced by the electrophoretic pattern shown in
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FIG. 2. Ascending electrophoretic patterns of DIP derivatives of crystalline chymotrypsins, in a sodium acetate buffer, pH 4.97, 0.1 ionic strength. Direction of migration from right to left, at 5.7 v. cm.+. Diagrams 14 represent the following conditions: (1) 2X crystallized DIP-cY-chymotrypsin; (9) DIP derivative of 2X crystallized &chymotrypsin; (5) 2~ crystallized DIP-r-chymotrypsin; (4) mixture of B part of DIP-B- and g of DIP-r-chymotrypsin. The times of electrophoresis were 1485, 1480, 1405, and 1350 min., respectively, for diagrams 14. The 6 boundary is not visible in the patterns due to countercurrent electrophoresis. The pattern at the bottom of diagram 4 is a representative Rayleigh fringe pattern.
Fig. 2, diagram 4, which has been obtained from a mixture containing N part of /3- and $5 part of y-DIP-chymotrypsin. Even upon prolonged electrophoresis (1350 min.) no resolution of the major peak could be observed. This finding is of particular interest since these two proteins have been found to have identical N- and C-terminal groups (15, 16). Sedimentation Figure 3 shows the concentration dependence of sedimentation constants of activation mixtures and crystalline proteins in buffered solutions of different pH. DIP-cY-chymotrypsin, at pH 3.86 (0.02 M sodium acetate, 0.18 M NaCl), shows the relatively high sedimentation constant which has been previously attributed to dimerization. However, the concentration dependence, though similar to that previously reported from this laboratory (16), is at variance with that described earlier by Schwert (l), since it shows a positive rather than a negative slope in the concentration range above 5 mg./ml. Similar sedimentation behavior was found for cY-chymotrypsin though variation among different preparations was observed. In contrast to these crystalline proteins, the DIP derivatives
ACTIVATION
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151
CHYMOTRYPSINOGEN pH 3.66 ACETATE
3.5
1
25J 3.5
l/
3.0
**
-
rt2.a2
*
Q 0 = ” 0 0
*
a
@
-
pH 7.5 PHOSPHATE w2. 0.1
PH 3.66
ACETATE
rf2’ 0.2
*
2.5-v
-
2.5 2.0
0
5.0
10.0 15.0 Protein Cont. mg./ ml.
20.0
25.0
dependence of sedimentation constants of chymotrypFIG. 3. Concentration sinogen, activation mixtures and crystalline chymotrypsins. The symbols refer to the following preparations: A chymotrypsinogen; @ rapid activation mixture containing /3-phenylpropionic acid (DIP-r-chymotrypsin) ; 0 rapid activation mixture (DIP-&chymotrypsin) ; q slow activation mixture, chymotrypsinogentrypsin, 5000:1, 28 hr. of incubation; q slow activation mixture [according to Kunita and Northrop (ll)]; 0 2X recrystallized DIP-a-chymotrypsin; q DIP derivative of 2 X crystallized ,%chymotrypsin.
of rapid as well as slow activation tation
constants
characteristic
mixtures4 revealed at this pH sedimen-
of that of a pure monomer (Fig. 3). How-
ever, a slow activation mixture prepared according to Kunitz and Norshowed a concentration dependence of throp [quoted in Ref. (ll)] sedimentation constants intermediate between that of chymotrypsinogen and DIP-a-chymotrypsin. After extrapolation to zero protein concentration, the sedimentation constant of DIP-&chymotrypsin wax Szo,W= 2.60. Only one sedimenting peak was seen in all patterns of the series represented by Fig. 3. It has been previously reported that the degree of dimerization of cr-chymotrypsin decreases with increasing pH within the pH range of 3.8-7.8 (1, 6). It was, therefore, of considerable interest to note that an activation mixture corresponding to DIP-T- and -6-chymotrypsin, respectively, while monomeric at pH 3.86, at pH 7.5 (K2HP04, 0.0308 M; KH~PO4,0.0072 M) revealed a concentration dependence characteristic 4 (chymotrypsinogen:trypsin,
5OOO:l)
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Fm. 4. Representative sedimentation patterns of DIP-chymotrypsins. (1) and (4) are rapid activation mixtures (DIP-khymotrypsins), at pH 3.86, 74 and 154 min., respectively, after reaching full speed; (2) and (6), the same preparation at pH 7.5, 74 and 146 min., respectively, after reaching full speed; (7) the same preparation at pH 3.0,70 min. after reaching full speed; (8) the same preparation in a synthetic b,oundary cell at pH 7.5,36 min. after reaching full speed. Protein concentration above synthetic boundary 12 mg./ml., and below the boundary, 16 mg./ml. (3) and (6) 2~ crystallized DIP-a-chymotrypsin, at pH 3.86,62 and 160 min. after reaching full speed. Protein concentration in runs 1,2,3 and 7 was 19,21, 17, and 20 mg./ml., respectively. The direction of sedimentation was from right to left.
of a monomer-dimer equilibrium (Fig. 3). These results were strictly reproducible; the observed sedimentation constants were independent of the time elapsed after preparation of the solutions (148 hr.). Furthermore, when a solution of DIP-&chymotrypsin, prepared at a high concentration, was diluted to a lower range, the sedimentation constant decreased in accordance with the plotted relation, demonstrating the ready reversibility of dimerization upon dilution. The pa-dependence of sedimentation constants of DIP-&chymotrypsin was also found to be strictly reversible. When a solution at pH 7.5 was equilibrated by dialysis against a pH 3.86 buffer and examined again in the ultracentrifuge, the results were identical with those obtained for solutions which were initially adjusted to pH 3.86. At pH 3.0 (0.02 M HCl, 0.08 M NaCl, titrated to pH
ACTIVATION OF CHYMOTRYP~INOGEN
153
3 with glycine) chymotrypsinogen, DIP derivatives of rapid and slow activation mixtures and crystalline DIP-W and -@-chymotrypsin are indistinguishable from one another in sedimentation behavior. The concentration dependence curve (Fig. 3) reveals the existence of a monomer throughout the entire concentration range studied. Thus a more reliable comparison of the sedimentation constants of chymotrypsinogen and the various chymotrypsins can be made, yielding a linearly extrapolated sedimentation constant of ~920,~= 2.46 S. This lower value, as compared to those found at the higher pH’s, may be due to the high charge on the protein. Representative sedimentation patterns are shown in Fig. 4. It will be noted that when the sedimentation constant shows a concentrat,ion dependence characteristic of a monomer-dimer equilibrium, the sedimenting boundary is distinctly asymmetric, with a trailing edge toward the solvent side. This is particularly evident in runs extended for longer periods of time (Fig. 4, patterns 5 and 6), but is also apparent when shorter term runs of DIP-&chymotrypsin at pH 7.5 (patterns 2 and 8) or DIP-a-chymotrypsin at 3.86 (pattern 3) are compared with DIP-& chymotrypsin at pH 3.86 or pH 3.0 (patterns 1,4, and 7). DIP-cu-chymotrypsin, which is also monomeric at pH 3.0, gave also a symmetrical boundary pattern at that pH. Included in Fig. 4 is a pattern obtained on DIP-d-chymotrypsin, pH 7.5, with the use of a synthetic boundary cell (17) (pattern 8). Because of the short duration of the run, the asymmetry to be expected for the boundary between protein and buffer is just barely noticeable, whereas, in accordance with expectation, no asymmetry is discernible in the synthetic boundary where protein concentration is relatively high on both sides of the boundary and is in a region where the slope of the concentration dependence curve is relatively small.
DISCUSSION The electrophoretic heterogeneity of crystalline a-chymotrypsin renders the interpretation of previous physical studies of this protein of doubtful significance. In contrast, the DIP derivatives of the major enzymatic components of rapid activation mixtures, i.e., rr- and d-chymotrypsins, were relatively homogeneous in electrophoresis. However, the heterogeneity increased as the time of activation increased and its rate decreased, probably due to secondary proteolytic degradation of the enzymes. It is of interest to note that the more heterogeneous activation mixtures do not readily lend themselves to fractionation by repeated crystallization.
154
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As evidenced by the data given in Table I, activation is accompanied by changes in electrophoretic mobility. The largest change, occurring during the conversion of r-chymotrypsin to the &form, can now be clearly ascribed to the liberation of the basic dipeptide, serylarginine (9). The mobility of the major protein component remains constant through the succeeding stages of activation leading to the formation of a-chymotrypsin. The subsequent shift to somewhat higher mobilities attending the formation of @-and y-chymotrypsin may be related to the release of a peptide having predominantly acidic properties. There is disconcerting disagreement among various investigators concerning the sedimentation behavior of crystalline cu-chymotrypsin (l-3, 6, 16). The most that can be said is that the exact shape of the curve relating sedimentation constants to protein concentration, though not clearly defined, varies with pH, the highest sedimentation constant being observed at about pH 3.86. In contrast, chymotrypsinogen, d-chymotrypsin, and relatively slow activation mixtures (28 hr. of activation, chymotrypsinogen-trypsin mole ratio 5000: 1) all reveal at pH 3.86 a sedimentation pattern characteristic of a monomer. The common feature of these proteins is that they are relatively homogeneous in electrophoresis and contain little, if any, @-or y-chymotrypsin. The slower, and more heterogeneous activation mixtures, prepared according to Kunitz and Northrop (1 l), contain more /3- or y-chymotrypsins and their sedimentation behavior resembles more nearly that of the crystalline (Y- and /3forms (Fig. 1). Thus the relative component distribution of a preparation appears to have a profound influence on its sedimentation behavior. In more alkaline solutions, i.e., pH 7.5, dimerization of cr-chymotrypsin decreases, whereas that of ?r- and Schymotrypsin increases. Chymotrypsinogen, however, remains as a monomer regardless of pH. It is, therefore, probable that the appearance of new terminal groups during activation is functionally related to the ability of the protein to dimerize. Since each of the enzymes appears to have its own characteristic pH dependence of dimerization, no generalization concerning the groups responsible for this process can yet be made. s However, since in terms of end-group analysis (7) and electrophoresis, ?r- and &chymotrypsins are 6 Although, after removal of the two C-terminal groups of DIP-a-chymotrypsin by carboxypeptidase the protein no longer dimerizea at pH 3.86 (16), the sedimentation constant increases with increasing protein concentration at pH 5.0, and more so at pH 6.2 (unpublished observations by Dr. J. Kraut). Since no electrophoretic patterns of these preparations are available, further interpretation of these findings is not warranted.
ACTIVATION
OF CHYMOTRYPSINOGEN
155
more nearly homogeneous than the crystalline (Y- and ,&forms (15, IS), the difference in sedimentation behavior between chymotrypsinogen and ?r-chymotrypsin appears to be more significant for the study of the activation process than is the difference between 6- and cY-chymotrypsins. In the light of recent work on the nature of the chemical changes accompanying the activation of chymotrypsinogen (7, 9), it is clear that the molecular weights of chymotrypsinogen and of r- and I-chymotrypsins are nearly identical, chymotrypsinogen differing from ?r- and 6chymotrypsin by 18 and 279, respectively. Hence, in view of the virtual identity of the extrapolated sedimentation constants at pH 3, the change in frictional ratios accompanying activation must be likewise negligibly small. Furthermore, since the extrapolated sedimentation constants of (Y- and @-chymotrypsins are indistinguishable from those of the above proteins, it seems probable that the proteolytic degradation attending the formation of the crystalline enzymes does not result in a large change in molecular weight. SUMMARY
Electrophoretic and ultracentrifugal measurements of chymotrypsinogen, of rapid and slow activation mixtures, and of crystalline chymotrypsins are described. Whereas chymotrypsinogen and rapid activation mixtures show a relatively high degree of electrophoretic homogeneity, the electrophoretic patterns become more complex and heterogeneous as the time of activation increases and the rate decreases. The DIP derivatives of crystalline, (Y-, fl-, and y-chymotrypsins are among the most heterogeneous products of this series of proteins. All of these proteins exist as a monomer at pH 3, within the concentration range of 2-20 mg./ml. In contrast to chymotrypsinogen, which is monomeric over the entire pH range studied, DIP-r- and -b-chymotrypsins at pH 7.5 exist in concentration-dependent monomer-dimer equilibrium. The pH dependence of dimerization of the crystalline enzymes differs markedly from those of the above proteins. REFERENCES 1. SCAVERT, G. W., J. Biol. Chem. 179,655 (1949). 2. S~HWERT, G. W., AND KAUFMAN, S., J. Biol. Chem. 190,799,807 (1951). 3. SMITH, E. L., AND BROWN, D. M., J. Biol. Chem. 196, 525 (1952). 4. EOAN, R., Federation Proc. 12, 199 (1953); 14, 206 (1955). 5. EGAN, R., MICHEL, H. O., AND SCXLUETER, R., Federation Proc. 13,202 (1954). 6. STEINER, R., Arch. Biochem. and Biophys. 63, 457 (1954).
156
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NEURATH
7. BETTELHEIM, F. R., AND NEURATH, H., J. Biol. Chem. 212,241 (1955). 8. DAVIE, E. W., AND NEURATH, H., J. Biol. Chem. 212, 515 (1955). 9. DREYER, W. J., AND NEURATH, H., J. Am. Chem. Sot. 77, 814 (1955); J. Biol. Chem. in press. 10. SVEDBERG, T., AND PEDERSEN, K. O., “The Ultracentrifuge.” Oxford University Press, Oxford, 1940. 11. NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., “Crystalline Enzymes.” Columbia University Press, New York, 1948. 12. KUNITZ, M., J. Gen. Physiol. 32, 265 (1948). 13. JACOBSEN, C. F., Compt. rend. trau. lab. Carlsberg, SCr. chim. 26, 325 (1947). (N eurath, H. and Bailey, K., eds.), Vol. 14. ALBERTY, R. A., in “The Proteins,” I, Chap. 6. Academic Press, New York, 1953. 15. ROVERY, C., FABRE, C., AND DESNUELLE, P., Biochim. et Biophys. Acta 10, 481 (1953). 16. GLADNER, J. A., AND NEURATH, H., J. Biol. Chem. 206,911 (1954). 17. PICKELS, E. G., HARRIN~TON, W. F., AND SCHACHMAN, H. K., Proc. Natl. Acad. Sci. U. S. 36,943 (1952).