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[90] Susceptibility to attack by proteolytic enzymes
[90]
SUSCEPTIBILITY TO PROamOI,YSlS
905
only when these are comparatively large, as in the case of parathyroid hormone C, or ribonuclease. Where such changes occur, the d a t a from thin-film dialysis can suggest the optimum solvent to make gel filtration more Selective. For example, the escape patterns ~1 for parathyroid hormone C in 0.01 N acetic acid, 0.3N acetic acid, and 0.2 M ammonium aceta~e-0.3N acetic acid are shown in Fig. 17. Gel filtration with Sephadex G-25 in 0.01 N acetic acid gave the pattern shown in Fig. 18; in 0 . 3 N acetic acid and 0 . 2 M ammonium a c e t a t e - 0 . 3 N acetic acid, Sephadex G-50 was required, as also shown in Fig. 18. II. Rasmussen and L. C. Craig, Biochim. Biophys. Acta ~ , 332 (1962).
[90] Susceptibility to Attack by Proteolytic
Enzymes
B y J. A. RUPLEY
Introduction I t has been recognized for several decades t h a t the rate of proteolysis depends upon the conformation of the protein substrate, 1 behavior most elegantly demonstrated by work at the Carlsberg Laboratory. 2-5 Proteolytic enzymes were first systematically employed to probe the fine structure of their substrates by Harrington, yon Hippel, and Mihalyi, e-8 who took advantage of the relatively rapid hydrolysis of exposed peptide bonds to estimate their number in native myosin and collagen. The following statements briefly summarize the conclusions about protein conformation that may be derived from the use of proteolytic enzymes: (1) The fraction of the peptide bonds of a w o t e i n which are exposed to the environment. This number should be approximately equal to the 1N. M. Green and H. Neurath, in "The Proteins" (H. Neurath and K. Bailey, eds.), 1st ed., Vol. II, Part B, p. 1057. Academic Press, New York, 1954. The section on proteolysis (p. 1171) is a thorough treatment of this subject, and is still relevant. K. LinderstrcSm-Lang and C. F. Jacobsen, Compt. Rend. Tray. Lab. Carlsberg, SeT. chim. 24, 1 (1941). s L. K. Christensen, Compt. Rend. Tray. Lab. Carlsberg, SeT. Chim. 28, 37 (1952). ' K. Linderstr0m-Lang, "Proteins and Enzymes," Lane Med. Lect., Vol. 6. Stanford Univ. Press, Stanford, California, 1952. s K . LinderstrCm-Lang, "Degradation of Proteins by Enzymes," Abstr. Proc. 9th Solvay Congr., 1953, p. 1. Much of this contribution is concerned with a theoretical treatment of proteolysis, which is illuminating but has not yet found application. *W. F. Harrington, P. H. yon Hippel, and E. Mihalyi, Bioehim. Biophys. Acta 32, 303 (1959). P. H. von Hippel and W. F. ttarrington, Biochim. Biophys. Acta 36, 427 (1959). E. Mihalyi and W. F. Harrington, Biochim. Biophys. Acta 36, 447 (1959).
906
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
fraction of all the peptide bonds compatible with the specificity of a given protease which are also accessible to the enzyme in the folded molecule. (2) Changes in confarmation. Changes in rate of proteolysis will reflect those changes in conformation which alter the number of accessible peptide bonds. (3) Location of regions of the polypeptide chain which are involved in conformational changes ar are exposed in the native molecule. Experiments with this objective are reasonable in view of the high rate of proteolysis of exposed bonds, which in favorable instances leads to the formation of isolatable intermediates exhibiting cleavages only in the exposed regions. Studies of this kind yield more detailed information than the foregoing, but are correspondingly more difficult and depend for interpretation upon knowledge of the amino acid sequence. Principles The structure of a protein is currently pictured as compact, with the polypeptide chain folded in a unique and highly organized fashion. The folding, however, can be largely irregular (i.e., of low helicity), and may be flexible in that the native molecule may exist as a set of closely related structures in mobile equilibrium2 This last point has no~ been clearly demonstrated, but witch its assumption a number of otherwise perplexing properties of proteins can be explained,x° The rate of proteolytic cleavage of a given peptide bond depends primarily upon two factors: first, the specificity of the protease, which restricts the rate of hydrolysis according to both the amino acids which contribute to the bond and to a lesser extent their near neighbors in the polypeptide chain; second, the accessibility of the bond, in that burial within the protein structure will hinder or completely prevent its cleavage. The second point is the one of interest for the present discus~J. A. Schellman and C. Schellman, in "The Proteins" (H. Neurath, ed.), 2rid ed., Vol. II, p. 1. Academic Press, New York, 1964. 10Flexibility may be particularly important for proteolysis. Only a ~raction of the total number of peptide bonds are on the surface of the molecule, and of these probably only a few are properly arranged so as to be accessible to an attacking enzyme. Indeed, a compact conformation conceivably may possess no bond which can be cleaved. In such instances, the observed rate of proteolysis may result entirely from attack upon bonds which are exposed in some partially unfolded species, present in low concentration and in equilibrium with the compact form (in effect, attack upon a "denatured" species). Since this problem has no immediate prospect of experimental treatment, it is perhaps best to beg the question by defining a protein species as a possible equilibrium mixture, involving a number of related forms.
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
907
sion, and the enzymic specificity will be considered only to the extent that correction for it is necessary.
Caurse of the Prot~olysis The initial event in the proteolysis of a native protein must take place at either a peptide bond exposed in a rigid, compact structure or one exposed in some partially unfolded species, a member of an equilibrium set characteristic of the native molecule. Cleavage of a small number of peptide bonds generally destabilizes the protein structure, leading to unfolding, exposure of more bonds to proteolytic action, and extensive degradation to small peptides. If this second phase of the proteolysis is much faster than the initial cleavage, then a molecule once attacked will be rapidly degraded to small peptides, and at any stage of the proteolysis only native protein and the products of extensive degradation will be present. If the initial attack is of a rate much greater than the subsequent cleavages, then all the reactant will be converted to a high-molecularweight intermediate, which may then degrade more or less slowly to limit peptides. Linderstr¢m-Lang ~ has termed these extreme cases, respectively, oneby-one and zipper types of reaction. Figure 1 shows for the two reaction types the expected change in some property peculiar to the native protein (e.g., enzymic activity or optical rotation) as a function of the
One-by-one type
Zipper-type F1o. I. The change in some property of the protein is shown as a function of the number of peptide bonds cleaved for the two extreme types of proteolytic reaction [from K. LinderstrCm-Lang, "Proteins and Enzymes," Lane Med. Lect., Vol. 6, Stanford Univ. Press, Stanford, California, 1952].
908
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
n u m b e r of peptide bonds split. Viewing experimental d a t a in this way, despite the simplicity of the approach, m a y substantially aid in understanding the kinetics of the reaction. One-by-one a t t a c k is observed for m a n y native proteins, in which all bonds are relatively resistant to proteolysis but cleavage of a few produces appreciable unfolding. Zipper a t t a c k 11 is characteristic of zymogen activation, 12 the formation of ribonuclease S, 13,~' and other reactions classed as limited proteolysis, in which a stable derivative is formed. Behavior intermediate between the two extremes has been frequently observed, for example in the proteolysis of t h e r m a l l y unfolded ribonuclease. ~5,16
Kinetic Scheme Describing the Events o] Proteoly~is The foregoing qualitative discussion can be summarized in the kinetic scheme shown in Fig. 2,1~ related to several previous proposals ~,ls and based on the discussion of I,inderstrCm-Lang.' There are equilibria between native (N~,o) and unfolded (D~,o) forms with equilibrium constants K~,.o. T h e concentration ofD~,o, but not its structure, is affected by the conditions of the reaction. Proteolysis of a given bond proceeds with a rate characterized b y the constant k~,~. This is large or small depending upon whether the bond is exposed or buried, and therefore conformational equilibria m a y determine the r a t e of hydrolysis. F o r the a r g u m e n t t h a t follows in the next section, the constants ko,~ and k~,~ a p p l y to exposed bonds in unfolded for.ms, and the constants k~,o to bonds in the native protein which m a y be either exposed or buried. T h e contribution of protease specificity to the rate is often assumed to be less i m p o r t a n t t h a n the conformational contribution (i.e., all k~,j for exposed bonds are assumed approximately equal). This point deserves 11The basis of the term "zipper" deserves comment. In LinderstrCm-Lang's words, "The enzyme may act in such a way that it runs from molecule to molecule and breaks, for instance, one peptide bond in each before it starts to cleave another bond in each, etc." It is not entirely clear whether Lang meant the word "zipper" to refer to the zipping about of the enzyme, or to the unzipping effect that cleavage of one bond often has on the protein structure. " H . Neurath, Advan. Protein Chem. 12, 320 (1957). "F. M. Richards, Compt. Rend. Tray. Lab. Carlsberg, Set. Chim. 29, 329 (1955). 1, F. M. Richards and P. J. Vithayathil, J. Biol. Chem. 234, 1459 (1959). ~J. A. Rupley and H. A. Scheraga, Biochemistry 2, 421 (1963). 16T. Ooi, J. A. Rupley, and H. A. Scheraga, Biochemistry 2, 432 (1963). 1~The kinetic scheme of Fig. 2 is capable of adjustment to meet specific cases, and is put forth in this special form as a basis for the discussion which follows. To generalize the scheme, it should be noted that conformational equilibria may involve all species, i.e., ones other than N,. o, and any species may participate in more than one equilibrium or cleavage reaction. H. A. Scheraga and J. A. Rupley, Advan. Enzymol. 24, 161 (1962).
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
koj, I
KNo,o N o, o ~
l
ko; ~-- Do, ~
Do, o
909
~ Do,z
.-~ etc.
~ DI, ~
>- etc.
kl, o
kl,1
K NI, o
N,,o ~
Ds,o
r-- D1, ~
kl,2
k2~, o N 2 , o - -
etc.
etc.
Fro. 2. A kinetic scheme that accounts for the course of proteolysis. comment, in view of the variation in rate of cleavage observed for even the same bond located in different sequences. 1 The difficulty can be neglected if an experimert is designed only to detect changes in conformation through changes in rate of proteolysis. However, the problem cannot be avoided in experiments designed to define thenumber of bonds exposed in a native protein or a partially unfolded species. Accessible bonds will be revealed in accordance with their rate of hydrolysis, and any estimate based on the kinetics of the proteolysis must give a lower limit on the number exposed. Mihalyi and Harrington 8 have remarked that the importance of the specificity contribution to the proteolytic process can be investigated by comparing the action of several enzymes on the same substrate. In the few cases for which this has been done, myosin8 and ribonuclease,15,18 the enzymic specificity did not affect the general conclusions reached about the protein structure; the results obtained with different proteases were consistent in terms of the region§ exposed, although different numbers and types of bonds were cleaved. Finally, for proteins the sequence of which has been determined with the aid of proteolytic fragmentation, assessment of the contribution of specificity to the cleavage rates can be approximated through considering the yields of the various peptides.
Interpretation o] the Kinetics o] Proteolysis in Terms of the Protein Structure (1) The rate constants ki, o will be large if the bonds to which they correspond are exposed in the native molecule. If the experimental condi-
910
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
tions favor the fully folded forms N~, o, then the hydrolysis of bonds other than those exposed will be slow, even if the constants k,,~ (for denatured species) are of the same magnitude as the k~,o (for the folded molecule). The two sets of reactions (corresponding to k~,o and k~,j) are then kinetically distinguishable, allowing estimation of the number of bonds in each class, namely those exposed and not exposed in the native protein. Isolation of the intermediates N,,o is also possible in principle, and has often been accomplished; for example, in the conversion of myosin to the meromyosins/and ribonuclease to ribonuclease S.1~ If the equilibrium constant K~.. is large, then~ as the constants kl,~ apply to exposed bonds and are large, the kinetic isolation of bonds split in the native protein from those split in the unfolded species becomes di~ieult or impossible. This imposes a fundamental limitation on the use of the prot~olytie enzymes to determine quantitatively the number of bonds exposed in a native molecule, success being likely only if none is exposed, or if their cleavage does not disrupt the folding. (2) The use of proteolytic enzymes to detect a change in protein conformation is understood in terms of a change in the equilibrium constant KN0.°. The increase in concentration of the unfolded species D0,o results in a significantly greater rate of pro~olysis which reflects the greater number of accessible peptide bonds. This situation is found, for example, in the hydrolysis of ribonuclease by chymotrypsin~5 and trypsin. ~e Figure 3 shows the increase in rate obtained by a small shift in a conformational equilibrium/9 induced by a change in temperature from 40.3 ° to 43.6 °. |
!
I
3.C 40.3 ° ~ 2.C
.6 °
1.0
_ _ o ~
°~°
o~ 0 0
I
I
I00 200 Time of Reoction (Min)
I
300
FIG. 3. The effect of temperature on the rate of ehymotryptic hydrolysis of ribonuclease A. T h e moles of base per mole of protein required to m a i n t a i n constant p H are shown as a function of time. T h e temperature of the reaction mixture was initially held at 40.3°; after 20 minutes it was raised rapidly t o 43.6 ° [from J. A. Rupley a n d H. A. Scheraga, Biochemistry 2, 421 (1963)]. ~ J . A. Hermans, Jr. and H. A. Scheraga, J. Am. Chem. ~qoc. 83, 3283 (1981).
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
911
(3) The location of bonds exposed in the native protein or rendered so by a conformational change requires the isolation of large intermediates (i.e.,Ni,0 or I)o,~),which will accumulate only if subsequent proteolytic steps are sufficientlyslow. Conditions of this sort are exemplified in the cleavage by subtilisinI' of the Ala-Ser (20-21) bond in native ribonuclease. Here /cI,0is large, secondary proteolytic events are much slower, and there is nearly quantitative conversion of ribonuclease to its derivative ribonuclease S. A more complex situation obtains in the chymotryptic or tryptic hydrolysis of thermally unfolded ribonuclease1"'1~: /c~,0is small, but thermal unfolding permits attack on a number of newly exposed bonds, with the formation of several related but chromatographically separable high-molecular-weight intermediates (peaks III-V in Fig. 4; peak II is undigested ribonuclease). The simultaneous appearance of substantial quantitiesof peptides of low molecular weight (rapidly eluting components in Fig. 4) shows that secondary (a)
0.5 0.4
0.~ 0.2 0.1 il IIl
II Ill
I Ill
Illll
llllll
I II'llillllil
Ilia
li
03 0.2 00.I 0
I I I
I i I I I
I IIIIi
IIiII
Iii
Iiillllllllll
ill
i
0.6 0.5 0.4
O3 02 O.i _
,l,llllilllll 50
Ilillll I00
150
Jlllllalllllllllllll 200
250
300
I 350
400
Time (rain)
FIG. 4. Chromatographic analyses of tryptic digests of ribonuclease A after three stages of digestion at 60°: (a) short-time, (b) medium-time, and (c) exhaustive digestion. Ordinate: optical density of the e~uent after reaction with n;,hydrin. Abscl.ssa: effluent volume expressed on a time scale for which 0~32 ml corresponds to 1 minute [from T. 0oi, J. A. Rupley, and H. A. Scheraga, Biochemistry 2, 432
(1963)].
912
INVESTIGATION" OF CONFORMATIONAL CHANGES
[90]
prot~olytic events are important, and these are likely to have followed extensive unfolding of structures weakened through the opening of peptide bonds. Thus, the location in the isolated intermediates of bonds which had been split may include some in regions which do not unfold in the initial, limited eonformational change. In complex situations of this sort, the data should be interpreted cautiously, and emphasis placed on cleavages obtained in high yield. Methods
Measurement of the Rate o! Proteolysis A variety of methods are available for measuring changes in substrate during proteolysis. These have been summarized by Green and Neurath, I and can be classed as reflecting either changes in number of intact peptide bonds or changes in some overall property of the protein. At least one method of the former type must be applied if conclusions are to be drawn from the rate of proteolysis. The following two procedures are recommended and frequently used: (1) The pH-stat 2° measures the number of peptide bonds cleaved through monitoring the hydrolytic reaction, - - C 0 - - N H - - + H20 --* aa --COO- + (I - al) - - C 0 0 H
q- a,--NH~ -{- (1 -- a,) --Nit, + -I- (a, -{- ~n -- 1) H+ where a, and a2 represent, respectively, the fraction of the earboxyl and amino groups dissociated. The limitations of the method derive from the pK's of the two protetropic groups formed in the proteolysis, approximately 4 for the a-carboxyl and 7.5 for the a-amino group. 2x First, the pH at which the reaction is measured must be chosen so that either a, < 1 and a2 = 0 (pH < 5.5), or a, = 1 and a2 > 0 (pH > 6.5). This restriction offers little problem, since the pH optima of most proteases do not lie in the insensitive region. Second, a quantitative correlation between the number of bonds cleaved and the amount of titrant required to maintain constant pH supposes knowledge of the pK important in determining the factor ( a x - l - a 2 - 1), which can be obtained only through titration of the new end groups formed in the proteolysis. Fortunately, the absolute number of bonds is not always necessary information, in particular for detection of chan~es in rate of hydrolysis. (2) The increase in ninhydrin coloff2 reflects the proteolytic rate '* C. F. Jacobsen, J. L~onis, K. Linderstrcm-Lang, and M. Ottesen, Methods Biochem. Anal. 4, 171 (1957). *' J. Steinhardt and S. Beyehok, in "The Proteins" (H. Neurath, ed.), 2rid eel., Vol. II, p. 139. Academic Press, New York, 1964. n See Yol. III [76, 78].
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
913
through the increase in new amino-terminal groups. Although the ninhydrin method does not show the course of the reaction while the experiment is in progress, as does the pH-stat, it requires no unusual equipment, is simple and accurate, and suffers no problem of instrumental stability if long times of proteotysis (several days) are necessary. Changes in an overall property of the protein are of particular interest for clarifying the interpretation of the rate of peptide bond cleavagc. The following examples serve to illustrate this: (1) A loss of enzymic activity more rapid than the rate of cleavage suggests the production of high-molecular-weight intermediates which are inactive or are of reduced activity. 16 (2) The absence of a change in optical rotation after hydrolysis of a small number of bonds supports a conclusion that there is no conformational change associated with the initial proteolytic events2 (3) Chromatographic separation of reaction products can demonstrate the formation and subsequent degradation of intermediates of relatively high molecular weight. 15,18 (4) The rate of appearance of products of low molecular weight may be followed by an increase in material soluble in trichloroacetic acid. s Clearly, a variety of other methods (ultracentrifugation, 8,23.2. dilatometry, 2 viscosimetry, 8,23 etc.) can be and have been used to follow changes in the protein substrate.
Concentration and Choice o] Proteolytic Enzyme Typical conditions of hydrolysis are approximately 10 mg per milliliter substrate and 0.1 mg per milliliter enzyme. The substrate concentration must be sufficiently high to allow measurement of the proteolytic changes, and for most methods 10 mg per milliliter is appropriate. A low concentration of protease relative to substrate minimizes complications arising from autolysis; the precise concentration chosen for a given experiment will be determined in large part by the need for obtaining significant hydrolysis within a reasonable time (ordinarily several hours). If a detailed kinetic analysis of the rates of proteo]ysis is of interest, the concentration of active proteolytic enzyme, which may suffer large decreases owing to autolysis, should be measured throughout the experiment. 2s The loss of enzymic activity is particularly troublesome = A. Ginsburg and H. K. Schachman, J. Biol. Chem. 235, 115 (1960). C. B. Anfinsen, J. Biol. Chem. 221, 405 (1956). ~*Standard methods of analysis using synthetic substrates are di~ussed under the various sections of this treatise dealing with the individual proteases. Also, see footnotes 1 and 26. ~*P. D. Boyer, H. Lardy, and K. Myrbiick (eds.), "The Enzymes," Vol. IV. Academic
914
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
for reactions at higher temperatures. For example, in the digestion of ribonuclease, even though the substrate protects against autolysis, half the activity of chymotrypsin or trypsin is lost in less than 1 hour at 50 ° and in 20 minutes at 600,27 and several charges of enzyme must be added to obtain significant reaction. Also, if rates for different conditions of reaction are to be compared, appropriate corrections must be made for changes in proteolytic activity with temperature, pH, and similar parameters. The purity of the enzyme is important, especially if the experiment is designed to locate cleavages in intermediate products. Fortunately, the commercially available preparations suitable for sequence studies are sufficiently pure. 28 More likely to be ignored, but of at least equal importance, is homogeneity of the substrate. In this connection it is noteworthy that at room temperature chymotrypsin was found to rapidly cleave peptide bonds in a crystalline sample of ribonuclease to the extent of approximately 1 per mole of protein, and a much slower proteolytic reaction followed, suggesting that a stable intermediate had been formed.26 However, the initial rapid reaction was not observed with chromatographically prepared ribonuclease A, indicating that the course of reaction found with the crystalline material was misleading and resulted from extensive degradation of one of several impurities in the sample. The choice of protease depends on the objective of the experiment. An endopeptidase of wide specificity, such as pepsin or chymotrypsin, is most suitable for defining the general exposure of peptide bonds. Trypsin is likely to serve better for the production of intermediates of high molecular weight which can be isolated and characterized, owing to the narrower specificity of the enzyme and, consequently, the fewer derivatives formed. In the proteolysis of thermally unfolded ribonuclease, chymotrypsin yields a large and complex set of products, 15 while trypsin yields only several le and in these the sites of cleavage can be unambiguously located. Exopeptidases, such as carboxypeptidase and leucine aminopeptidase, can be used to determine the accessibility of the terminal regions of the polypeptide chain. For example, ribonuclease is resistant to carboxypeptidase action, but the derivative ribonuclease S readily loses its C-terminal valine. 2~ " J. A. Rupley and H. A. Scheraga, unpublished experiments. '* See this volume [24, 25, 47]. "~J. T. Potts, Jr., D. M. Young, C. B. Anfinsen, and A. Sandoval, J. Biol. Chem. 239, 3781 (1964). Press, New York, 1960. This volume includes reviews of the chemistry of various proteolytic enzymes.
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
915
Temperature The rate of proteolysis will vary strongly with temperature over ranges in which conformational changes in the substrate occur (see Fig. 3), and maintenance of constant temperature is essential. If determination of bonds exposed in the native protein is of interest, the temperature of the experiment should be chosen to minimize unfolding of initial products. For example, cleavage by subtilisin of the Ala-Ser (20-21) bond in ribonuclease is better isolated from secondary events by working at 3 ° instead of 300. la,14 Alternatively, as has been discussed, the temperature dependence of proteolytic rates may be used to characterize thermal transitions in proteins25,~
pH The choice of pH is limited by the vaxiation with this parameter of both the proteolytic activity and the substrate conformation. Fortunately, proteases axe available which together span nearly the entire pH range that is of interest. ~ Trypsin and ehymotrypsin, with pH optima of about 8, are useful from pH 6 to above 10; pepsin, from pH 1 to above 2; and the plant proteases, papain and ficin, from pH less than 4 through to the alkaline range, pH-dependent conformational changes in the substrate may be avoided or studied, as with the corresponding changes produced by temperature.
Solvent The solvent, like pH and temperature, may affect both protease and substrate, and its composition must be controlled. Certain divalent ions, in particular calcium, stabilize trypsin solutions, 1,~ and metal ions such as manganese and magnesium activate leucine aminopeptidase. ~ Perhaps more significant for the present discussion are specific effects which probably involve the substrate. In the chymotryptic hydrolysis of thermally unfolded ribonuclease, the addition of calcium ion or phosphate ion, or increased ionic strength, altered the course of the proteolysis, in particular as regards the formation of intermediates of high molecular weight25 Nonaqueous solvents often induce conformational changes in proteins; in accord with this, the tryptic digestion of ribonuclease proceeds rapidly at 35 ° in the presence of 25% propanol, 8° while in contrast temperatures of about 60 ° are required for a comparable proteolytic rate in water. IoT. Ooi and H. A. Scheraga, Biochemistry 3, 1209 (1964).
916
INVESTIGATION OF CONFORMATIONAL CHANGES
[O0]
Analysis o] Kinetic Data In the detection of structural changes through the rate of proteolysis, qualitative comparison of the initial rates is frequently sufficient to indicate whether there is exposure and its approximate extent. In contrast, a quantitative assessment of the number of exposed bonds susceptible to cleavage requires a protein substrate which does not change conformation (the number of exposed bonds) over an extent of proteolysis sufficiently large to permit analysis of the initial stage of the reaction according to some standard kinetic treatment. The preceding discussion has indicated that this behavior can be expected only when relatively stable derivatives are formed. Harrington and co-workers7,s have investigated systems of this sort, the hydrolysis of myosin by trypsin and of collagen by collagenase. In these cases fast and slow reactions had sufficiently different rate constants that their kinetics could be studied independently. The fast reaction, which produced the meromyosins from myosin, was shown to have little effect on the folding of the molecule, and, in contrast, the subsequent slow reaction, in which 4 times as many bonds were cleaved, led to a significant conformational change. Location o] Bonds Hydrolyzed The study of derivatives of high molecular weight has been discussed, in preceding sections, with reference to defining exposed regions of the polypeptide chain. Although the techniques employed must be developed and will be different for each particular protein, certain comments are appropriate. Separation methods for protein mixtures have reached a high state of development, and although isolation of one reaction product from a complex mixture is difficult it is no longer an overwhelming problem21 The location of sites of cleavage is most fruitful for proteins the sequence of which is known or under study, and in such instances relatively simple analyses of the purified derivatives may be sufficient. For example, in the tryptic hydrolysis of thermally unfolded ribonuelease, cleavage at Lys-Ser (31-32) and Arg-Asp(NH2) (33-34) was established by using only amino acid and end-group analyses. 16 In more complex cases, methods of the sort employed in the original sequence determination become necessary23 *1See Vol. I [I1, 12, 13] and Vol. IV [1]; see also footnote 32. H. A. Sober, R. W. Hartley, Jr., W. R. Carroll, and E. A. Peterson, in "The Proteins" (H. Neurath, ed.), 2nd ed., Vol. III, p. 1. Academic Press, New York, 1965. u See the appropriate sections of this volume, and also footnote 34. R. E. Canfield and C. B. Anfinsen, in "The Proteins" (H. Neurath, ed.), 2nd ed., Vol. I, p. 311. Academic Press, New York, 1963.
[91]
IMMUNOLOGICAL
TECHNIQUES
917
Relationship to Other Methods ]or Investigation o] Con]ormational Changes The kinetic basis of this procedure differentiates it from equilibrium measurements such as titration or chromophore perturbation. In contrast, however, there is a strong similarity to hydrogen exchange,35,se which like susceptibility to proteolysis can reflect flexibility (motility) of the protein. Specifically, a partially unfolded or expanded state may be improbable and of no significance for equilibrium behavior, but so reactive that it dominates kinetic processes such as proteolysis or exchange (in Fig. 2, KNo.0 might be small, but ko,1 if sufficiently large could still dominate kl, o). A conformational change detected by an altered rate of proteolytic cleavage may represent (although not necessarily) an increase in the fraction of molecules in an expanded conformation, this fraction even after its increase being so small as to be unimportant for the equilibrium properties. An effect of this sort should be distinguished from one in which the average conformation is disorganized to a significant extent. It is of interest that the sensitivity of ribonuclease to chymotrypsin develops at temperatures in the lower part of the range for the transition determined by optical methods. 16,~9 Also, although a peptide bond of a native protein may be inaccessible in the most probable or in the average molecular conformation, in spite of this proteolysis could see the bond as "exposed" provided it had a high probability of exposure relative to other bonds. Questions of this sort should be resolved after data have been accumulated for crystallographically defined proteins, but until then, the foregoing broader understanding of protein conformation and conformational changes must be kept in mind when interpreting proteolytic reactions. See this volume [85]. A. Hvidt and S. O. Nielsen, Advan. Protein Chem. 21, 287 (1966).
[91] I m m u n o l o g i c a l T e c h n i q u e s ( G e n e r a l ) B y RAY K. BROWN
This chapter concerns: the preparation of antisera; general tests for identification of the immune system and of impurities; and the precipitin reaction. Careful studies require a variety of techniques. Complement fixation, a micro form of which is described in the following chapter, hemagglutination, and fluorescence are other important techniques. The inhibition of these reactions is often studied. Radioactive tracers are also useful, especially for systems with nonprecipitating antibody.
SUSCEPTIBILITY TO PROamOI,YSlS
905
only when these are comparatively large, as in the case of parathyroid hormone C, or ribonuclease. Where such changes occur, the d a t a from thin-film dialysis can suggest the optimum solvent to make gel filtration more Selective. For example, the escape patterns ~1 for parathyroid hormone C in 0.01 N acetic acid, 0.3N acetic acid, and 0.2 M ammonium aceta~e-0.3N acetic acid are shown in Fig. 17. Gel filtration with Sephadex G-25 in 0.01 N acetic acid gave the pattern shown in Fig. 18; in 0 . 3 N acetic acid and 0 . 2 M ammonium a c e t a t e - 0 . 3 N acetic acid, Sephadex G-50 was required, as also shown in Fig. 18. II. Rasmussen and L. C. Craig, Biochim. Biophys. Acta ~ , 332 (1962).
[90] Susceptibility to Attack by Proteolytic
Enzymes
B y J. A. RUPLEY
Introduction I t has been recognized for several decades t h a t the rate of proteolysis depends upon the conformation of the protein substrate, 1 behavior most elegantly demonstrated by work at the Carlsberg Laboratory. 2-5 Proteolytic enzymes were first systematically employed to probe the fine structure of their substrates by Harrington, yon Hippel, and Mihalyi, e-8 who took advantage of the relatively rapid hydrolysis of exposed peptide bonds to estimate their number in native myosin and collagen. The following statements briefly summarize the conclusions about protein conformation that may be derived from the use of proteolytic enzymes: (1) The fraction of the peptide bonds of a w o t e i n which are exposed to the environment. This number should be approximately equal to the 1N. M. Green and H. Neurath, in "The Proteins" (H. Neurath and K. Bailey, eds.), 1st ed., Vol. II, Part B, p. 1057. Academic Press, New York, 1954. The section on proteolysis (p. 1171) is a thorough treatment of this subject, and is still relevant. K. LinderstrcSm-Lang and C. F. Jacobsen, Compt. Rend. Tray. Lab. Carlsberg, SeT. chim. 24, 1 (1941). s L. K. Christensen, Compt. Rend. Tray. Lab. Carlsberg, SeT. Chim. 28, 37 (1952). ' K. Linderstr0m-Lang, "Proteins and Enzymes," Lane Med. Lect., Vol. 6. Stanford Univ. Press, Stanford, California, 1952. s K . LinderstrCm-Lang, "Degradation of Proteins by Enzymes," Abstr. Proc. 9th Solvay Congr., 1953, p. 1. Much of this contribution is concerned with a theoretical treatment of proteolysis, which is illuminating but has not yet found application. *W. F. Harrington, P. H. yon Hippel, and E. Mihalyi, Bioehim. Biophys. Acta 32, 303 (1959). P. H. von Hippel and W. F. ttarrington, Biochim. Biophys. Acta 36, 427 (1959). E. Mihalyi and W. F. Harrington, Biochim. Biophys. Acta 36, 447 (1959).
906
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
fraction of all the peptide bonds compatible with the specificity of a given protease which are also accessible to the enzyme in the folded molecule. (2) Changes in confarmation. Changes in rate of proteolysis will reflect those changes in conformation which alter the number of accessible peptide bonds. (3) Location of regions of the polypeptide chain which are involved in conformational changes ar are exposed in the native molecule. Experiments with this objective are reasonable in view of the high rate of proteolysis of exposed bonds, which in favorable instances leads to the formation of isolatable intermediates exhibiting cleavages only in the exposed regions. Studies of this kind yield more detailed information than the foregoing, but are correspondingly more difficult and depend for interpretation upon knowledge of the amino acid sequence. Principles The structure of a protein is currently pictured as compact, with the polypeptide chain folded in a unique and highly organized fashion. The folding, however, can be largely irregular (i.e., of low helicity), and may be flexible in that the native molecule may exist as a set of closely related structures in mobile equilibrium2 This last point has no~ been clearly demonstrated, but witch its assumption a number of otherwise perplexing properties of proteins can be explained,x° The rate of proteolytic cleavage of a given peptide bond depends primarily upon two factors: first, the specificity of the protease, which restricts the rate of hydrolysis according to both the amino acids which contribute to the bond and to a lesser extent their near neighbors in the polypeptide chain; second, the accessibility of the bond, in that burial within the protein structure will hinder or completely prevent its cleavage. The second point is the one of interest for the present discus~J. A. Schellman and C. Schellman, in "The Proteins" (H. Neurath, ed.), 2rid ed., Vol. II, p. 1. Academic Press, New York, 1964. 10Flexibility may be particularly important for proteolysis. Only a ~raction of the total number of peptide bonds are on the surface of the molecule, and of these probably only a few are properly arranged so as to be accessible to an attacking enzyme. Indeed, a compact conformation conceivably may possess no bond which can be cleaved. In such instances, the observed rate of proteolysis may result entirely from attack upon bonds which are exposed in some partially unfolded species, present in low concentration and in equilibrium with the compact form (in effect, attack upon a "denatured" species). Since this problem has no immediate prospect of experimental treatment, it is perhaps best to beg the question by defining a protein species as a possible equilibrium mixture, involving a number of related forms.
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
907
sion, and the enzymic specificity will be considered only to the extent that correction for it is necessary.
Caurse of the Prot~olysis The initial event in the proteolysis of a native protein must take place at either a peptide bond exposed in a rigid, compact structure or one exposed in some partially unfolded species, a member of an equilibrium set characteristic of the native molecule. Cleavage of a small number of peptide bonds generally destabilizes the protein structure, leading to unfolding, exposure of more bonds to proteolytic action, and extensive degradation to small peptides. If this second phase of the proteolysis is much faster than the initial cleavage, then a molecule once attacked will be rapidly degraded to small peptides, and at any stage of the proteolysis only native protein and the products of extensive degradation will be present. If the initial attack is of a rate much greater than the subsequent cleavages, then all the reactant will be converted to a high-molecularweight intermediate, which may then degrade more or less slowly to limit peptides. Linderstr¢m-Lang ~ has termed these extreme cases, respectively, oneby-one and zipper types of reaction. Figure 1 shows for the two reaction types the expected change in some property peculiar to the native protein (e.g., enzymic activity or optical rotation) as a function of the
One-by-one type
Zipper-type F1o. I. The change in some property of the protein is shown as a function of the number of peptide bonds cleaved for the two extreme types of proteolytic reaction [from K. LinderstrCm-Lang, "Proteins and Enzymes," Lane Med. Lect., Vol. 6, Stanford Univ. Press, Stanford, California, 1952].
908
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
n u m b e r of peptide bonds split. Viewing experimental d a t a in this way, despite the simplicity of the approach, m a y substantially aid in understanding the kinetics of the reaction. One-by-one a t t a c k is observed for m a n y native proteins, in which all bonds are relatively resistant to proteolysis but cleavage of a few produces appreciable unfolding. Zipper a t t a c k 11 is characteristic of zymogen activation, 12 the formation of ribonuclease S, 13,~' and other reactions classed as limited proteolysis, in which a stable derivative is formed. Behavior intermediate between the two extremes has been frequently observed, for example in the proteolysis of t h e r m a l l y unfolded ribonuclease. ~5,16
Kinetic Scheme Describing the Events o] Proteoly~is The foregoing qualitative discussion can be summarized in the kinetic scheme shown in Fig. 2,1~ related to several previous proposals ~,ls and based on the discussion of I,inderstrCm-Lang.' There are equilibria between native (N~,o) and unfolded (D~,o) forms with equilibrium constants K~,.o. T h e concentration ofD~,o, but not its structure, is affected by the conditions of the reaction. Proteolysis of a given bond proceeds with a rate characterized b y the constant k~,~. This is large or small depending upon whether the bond is exposed or buried, and therefore conformational equilibria m a y determine the r a t e of hydrolysis. F o r the a r g u m e n t t h a t follows in the next section, the constants ko,~ and k~,~ a p p l y to exposed bonds in unfolded for.ms, and the constants k~,o to bonds in the native protein which m a y be either exposed or buried. T h e contribution of protease specificity to the rate is often assumed to be less i m p o r t a n t t h a n the conformational contribution (i.e., all k~,j for exposed bonds are assumed approximately equal). This point deserves 11The basis of the term "zipper" deserves comment. In LinderstrCm-Lang's words, "The enzyme may act in such a way that it runs from molecule to molecule and breaks, for instance, one peptide bond in each before it starts to cleave another bond in each, etc." It is not entirely clear whether Lang meant the word "zipper" to refer to the zipping about of the enzyme, or to the unzipping effect that cleavage of one bond often has on the protein structure. " H . Neurath, Advan. Protein Chem. 12, 320 (1957). "F. M. Richards, Compt. Rend. Tray. Lab. Carlsberg, Set. Chim. 29, 329 (1955). 1, F. M. Richards and P. J. Vithayathil, J. Biol. Chem. 234, 1459 (1959). ~J. A. Rupley and H. A. Scheraga, Biochemistry 2, 421 (1963). 16T. Ooi, J. A. Rupley, and H. A. Scheraga, Biochemistry 2, 432 (1963). 1~The kinetic scheme of Fig. 2 is capable of adjustment to meet specific cases, and is put forth in this special form as a basis for the discussion which follows. To generalize the scheme, it should be noted that conformational equilibria may involve all species, i.e., ones other than N,. o, and any species may participate in more than one equilibrium or cleavage reaction. H. A. Scheraga and J. A. Rupley, Advan. Enzymol. 24, 161 (1962).
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
koj, I
KNo,o N o, o ~
l
ko; ~-- Do, ~
Do, o
909
~ Do,z
.-~ etc.
~ DI, ~
>- etc.
kl, o
kl,1
K NI, o
N,,o ~
Ds,o
r-- D1, ~
kl,2
k2~, o N 2 , o - -
etc.
etc.
Fro. 2. A kinetic scheme that accounts for the course of proteolysis. comment, in view of the variation in rate of cleavage observed for even the same bond located in different sequences. 1 The difficulty can be neglected if an experimert is designed only to detect changes in conformation through changes in rate of proteolysis. However, the problem cannot be avoided in experiments designed to define thenumber of bonds exposed in a native protein or a partially unfolded species. Accessible bonds will be revealed in accordance with their rate of hydrolysis, and any estimate based on the kinetics of the proteolysis must give a lower limit on the number exposed. Mihalyi and Harrington 8 have remarked that the importance of the specificity contribution to the proteolytic process can be investigated by comparing the action of several enzymes on the same substrate. In the few cases for which this has been done, myosin8 and ribonuclease,15,18 the enzymic specificity did not affect the general conclusions reached about the protein structure; the results obtained with different proteases were consistent in terms of the region§ exposed, although different numbers and types of bonds were cleaved. Finally, for proteins the sequence of which has been determined with the aid of proteolytic fragmentation, assessment of the contribution of specificity to the cleavage rates can be approximated through considering the yields of the various peptides.
Interpretation o] the Kinetics o] Proteolysis in Terms of the Protein Structure (1) The rate constants ki, o will be large if the bonds to which they correspond are exposed in the native molecule. If the experimental condi-
910
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
tions favor the fully folded forms N~, o, then the hydrolysis of bonds other than those exposed will be slow, even if the constants k,,~ (for denatured species) are of the same magnitude as the k~,o (for the folded molecule). The two sets of reactions (corresponding to k~,o and k~,j) are then kinetically distinguishable, allowing estimation of the number of bonds in each class, namely those exposed and not exposed in the native protein. Isolation of the intermediates N,,o is also possible in principle, and has often been accomplished; for example, in the conversion of myosin to the meromyosins/and ribonuclease to ribonuclease S.1~ If the equilibrium constant K~.. is large, then~ as the constants kl,~ apply to exposed bonds and are large, the kinetic isolation of bonds split in the native protein from those split in the unfolded species becomes di~ieult or impossible. This imposes a fundamental limitation on the use of the prot~olytie enzymes to determine quantitatively the number of bonds exposed in a native molecule, success being likely only if none is exposed, or if their cleavage does not disrupt the folding. (2) The use of proteolytic enzymes to detect a change in protein conformation is understood in terms of a change in the equilibrium constant KN0.°. The increase in concentration of the unfolded species D0,o results in a significantly greater rate of pro~olysis which reflects the greater number of accessible peptide bonds. This situation is found, for example, in the hydrolysis of ribonuclease by chymotrypsin~5 and trypsin. ~e Figure 3 shows the increase in rate obtained by a small shift in a conformational equilibrium/9 induced by a change in temperature from 40.3 ° to 43.6 °. |
!
I
3.C 40.3 ° ~ 2.C
.6 °
1.0
_ _ o ~
°~°
o~ 0 0
I
I
I00 200 Time of Reoction (Min)
I
300
FIG. 3. The effect of temperature on the rate of ehymotryptic hydrolysis of ribonuclease A. T h e moles of base per mole of protein required to m a i n t a i n constant p H are shown as a function of time. T h e temperature of the reaction mixture was initially held at 40.3°; after 20 minutes it was raised rapidly t o 43.6 ° [from J. A. Rupley a n d H. A. Scheraga, Biochemistry 2, 421 (1963)]. ~ J . A. Hermans, Jr. and H. A. Scheraga, J. Am. Chem. ~qoc. 83, 3283 (1981).
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
911
(3) The location of bonds exposed in the native protein or rendered so by a conformational change requires the isolation of large intermediates (i.e.,Ni,0 or I)o,~),which will accumulate only if subsequent proteolytic steps are sufficientlyslow. Conditions of this sort are exemplified in the cleavage by subtilisinI' of the Ala-Ser (20-21) bond in native ribonuclease. Here /cI,0is large, secondary proteolytic events are much slower, and there is nearly quantitative conversion of ribonuclease to its derivative ribonuclease S. A more complex situation obtains in the chymotryptic or tryptic hydrolysis of thermally unfolded ribonuclease1"'1~: /c~,0is small, but thermal unfolding permits attack on a number of newly exposed bonds, with the formation of several related but chromatographically separable high-molecular-weight intermediates (peaks III-V in Fig. 4; peak II is undigested ribonuclease). The simultaneous appearance of substantial quantitiesof peptides of low molecular weight (rapidly eluting components in Fig. 4) shows that secondary (a)
0.5 0.4
0.~ 0.2 0.1 il IIl
II Ill
I Ill
Illll
llllll
I II'llillllil
Ilia
li
03 0.2 00.I 0
I I I
I i I I I
I IIIIi
IIiII
Iii
Iiillllllllll
ill
i
0.6 0.5 0.4
O3 02 O.i _
,l,llllilllll 50
Ilillll I00
150
Jlllllalllllllllllll 200
250
300
I 350
400
Time (rain)
FIG. 4. Chromatographic analyses of tryptic digests of ribonuclease A after three stages of digestion at 60°: (a) short-time, (b) medium-time, and (c) exhaustive digestion. Ordinate: optical density of the e~uent after reaction with n;,hydrin. Abscl.ssa: effluent volume expressed on a time scale for which 0~32 ml corresponds to 1 minute [from T. 0oi, J. A. Rupley, and H. A. Scheraga, Biochemistry 2, 432
(1963)].
912
INVESTIGATION" OF CONFORMATIONAL CHANGES
[90]
prot~olytic events are important, and these are likely to have followed extensive unfolding of structures weakened through the opening of peptide bonds. Thus, the location in the isolated intermediates of bonds which had been split may include some in regions which do not unfold in the initial, limited eonformational change. In complex situations of this sort, the data should be interpreted cautiously, and emphasis placed on cleavages obtained in high yield. Methods
Measurement of the Rate o! Proteolysis A variety of methods are available for measuring changes in substrate during proteolysis. These have been summarized by Green and Neurath, I and can be classed as reflecting either changes in number of intact peptide bonds or changes in some overall property of the protein. At least one method of the former type must be applied if conclusions are to be drawn from the rate of proteolysis. The following two procedures are recommended and frequently used: (1) The pH-stat 2° measures the number of peptide bonds cleaved through monitoring the hydrolytic reaction, - - C 0 - - N H - - + H20 --* aa --COO- + (I - al) - - C 0 0 H
q- a,--NH~ -{- (1 -- a,) --Nit, + -I- (a, -{- ~n -- 1) H+ where a, and a2 represent, respectively, the fraction of the earboxyl and amino groups dissociated. The limitations of the method derive from the pK's of the two protetropic groups formed in the proteolysis, approximately 4 for the a-carboxyl and 7.5 for the a-amino group. 2x First, the pH at which the reaction is measured must be chosen so that either a, < 1 and a2 = 0 (pH < 5.5), or a, = 1 and a2 > 0 (pH > 6.5). This restriction offers little problem, since the pH optima of most proteases do not lie in the insensitive region. Second, a quantitative correlation between the number of bonds cleaved and the amount of titrant required to maintain constant pH supposes knowledge of the pK important in determining the factor ( a x - l - a 2 - 1), which can be obtained only through titration of the new end groups formed in the proteolysis. Fortunately, the absolute number of bonds is not always necessary information, in particular for detection of chan~es in rate of hydrolysis. (2) The increase in ninhydrin coloff2 reflects the proteolytic rate '* C. F. Jacobsen, J. L~onis, K. Linderstrcm-Lang, and M. Ottesen, Methods Biochem. Anal. 4, 171 (1957). *' J. Steinhardt and S. Beyehok, in "The Proteins" (H. Neurath, ed.), 2rid eel., Vol. II, p. 139. Academic Press, New York, 1964. n See Yol. III [76, 78].
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
913
through the increase in new amino-terminal groups. Although the ninhydrin method does not show the course of the reaction while the experiment is in progress, as does the pH-stat, it requires no unusual equipment, is simple and accurate, and suffers no problem of instrumental stability if long times of proteotysis (several days) are necessary. Changes in an overall property of the protein are of particular interest for clarifying the interpretation of the rate of peptide bond cleavagc. The following examples serve to illustrate this: (1) A loss of enzymic activity more rapid than the rate of cleavage suggests the production of high-molecular-weight intermediates which are inactive or are of reduced activity. 16 (2) The absence of a change in optical rotation after hydrolysis of a small number of bonds supports a conclusion that there is no conformational change associated with the initial proteolytic events2 (3) Chromatographic separation of reaction products can demonstrate the formation and subsequent degradation of intermediates of relatively high molecular weight. 15,18 (4) The rate of appearance of products of low molecular weight may be followed by an increase in material soluble in trichloroacetic acid. s Clearly, a variety of other methods (ultracentrifugation, 8,23.2. dilatometry, 2 viscosimetry, 8,23 etc.) can be and have been used to follow changes in the protein substrate.
Concentration and Choice o] Proteolytic Enzyme Typical conditions of hydrolysis are approximately 10 mg per milliliter substrate and 0.1 mg per milliliter enzyme. The substrate concentration must be sufficiently high to allow measurement of the proteolytic changes, and for most methods 10 mg per milliliter is appropriate. A low concentration of protease relative to substrate minimizes complications arising from autolysis; the precise concentration chosen for a given experiment will be determined in large part by the need for obtaining significant hydrolysis within a reasonable time (ordinarily several hours). If a detailed kinetic analysis of the rates of proteo]ysis is of interest, the concentration of active proteolytic enzyme, which may suffer large decreases owing to autolysis, should be measured throughout the experiment. 2s The loss of enzymic activity is particularly troublesome = A. Ginsburg and H. K. Schachman, J. Biol. Chem. 235, 115 (1960). C. B. Anfinsen, J. Biol. Chem. 221, 405 (1956). ~*Standard methods of analysis using synthetic substrates are di~ussed under the various sections of this treatise dealing with the individual proteases. Also, see footnotes 1 and 26. ~*P. D. Boyer, H. Lardy, and K. Myrbiick (eds.), "The Enzymes," Vol. IV. Academic
914
INVESTIGATION OF CONFORMATIONAL CHANGES
[90]
for reactions at higher temperatures. For example, in the digestion of ribonuclease, even though the substrate protects against autolysis, half the activity of chymotrypsin or trypsin is lost in less than 1 hour at 50 ° and in 20 minutes at 600,27 and several charges of enzyme must be added to obtain significant reaction. Also, if rates for different conditions of reaction are to be compared, appropriate corrections must be made for changes in proteolytic activity with temperature, pH, and similar parameters. The purity of the enzyme is important, especially if the experiment is designed to locate cleavages in intermediate products. Fortunately, the commercially available preparations suitable for sequence studies are sufficiently pure. 28 More likely to be ignored, but of at least equal importance, is homogeneity of the substrate. In this connection it is noteworthy that at room temperature chymotrypsin was found to rapidly cleave peptide bonds in a crystalline sample of ribonuclease to the extent of approximately 1 per mole of protein, and a much slower proteolytic reaction followed, suggesting that a stable intermediate had been formed.26 However, the initial rapid reaction was not observed with chromatographically prepared ribonuclease A, indicating that the course of reaction found with the crystalline material was misleading and resulted from extensive degradation of one of several impurities in the sample. The choice of protease depends on the objective of the experiment. An endopeptidase of wide specificity, such as pepsin or chymotrypsin, is most suitable for defining the general exposure of peptide bonds. Trypsin is likely to serve better for the production of intermediates of high molecular weight which can be isolated and characterized, owing to the narrower specificity of the enzyme and, consequently, the fewer derivatives formed. In the proteolysis of thermally unfolded ribonuclease, chymotrypsin yields a large and complex set of products, 15 while trypsin yields only several le and in these the sites of cleavage can be unambiguously located. Exopeptidases, such as carboxypeptidase and leucine aminopeptidase, can be used to determine the accessibility of the terminal regions of the polypeptide chain. For example, ribonuclease is resistant to carboxypeptidase action, but the derivative ribonuclease S readily loses its C-terminal valine. 2~ " J. A. Rupley and H. A. Scheraga, unpublished experiments. '* See this volume [24, 25, 47]. "~J. T. Potts, Jr., D. M. Young, C. B. Anfinsen, and A. Sandoval, J. Biol. Chem. 239, 3781 (1964). Press, New York, 1960. This volume includes reviews of the chemistry of various proteolytic enzymes.
[90]
SUSCEPTIBILITY TO PROTEOLYSIS
915
Temperature The rate of proteolysis will vary strongly with temperature over ranges in which conformational changes in the substrate occur (see Fig. 3), and maintenance of constant temperature is essential. If determination of bonds exposed in the native protein is of interest, the temperature of the experiment should be chosen to minimize unfolding of initial products. For example, cleavage by subtilisin of the Ala-Ser (20-21) bond in ribonuclease is better isolated from secondary events by working at 3 ° instead of 300. la,14 Alternatively, as has been discussed, the temperature dependence of proteolytic rates may be used to characterize thermal transitions in proteins25,~
pH The choice of pH is limited by the vaxiation with this parameter of both the proteolytic activity and the substrate conformation. Fortunately, proteases axe available which together span nearly the entire pH range that is of interest. ~ Trypsin and ehymotrypsin, with pH optima of about 8, are useful from pH 6 to above 10; pepsin, from pH 1 to above 2; and the plant proteases, papain and ficin, from pH less than 4 through to the alkaline range, pH-dependent conformational changes in the substrate may be avoided or studied, as with the corresponding changes produced by temperature.
Solvent The solvent, like pH and temperature, may affect both protease and substrate, and its composition must be controlled. Certain divalent ions, in particular calcium, stabilize trypsin solutions, 1,~ and metal ions such as manganese and magnesium activate leucine aminopeptidase. ~ Perhaps more significant for the present discussion are specific effects which probably involve the substrate. In the chymotryptic hydrolysis of thermally unfolded ribonuclease, the addition of calcium ion or phosphate ion, or increased ionic strength, altered the course of the proteolysis, in particular as regards the formation of intermediates of high molecular weight25 Nonaqueous solvents often induce conformational changes in proteins; in accord with this, the tryptic digestion of ribonuclease proceeds rapidly at 35 ° in the presence of 25% propanol, 8° while in contrast temperatures of about 60 ° are required for a comparable proteolytic rate in water. IoT. Ooi and H. A. Scheraga, Biochemistry 3, 1209 (1964).
916
INVESTIGATION OF CONFORMATIONAL CHANGES
[O0]
Analysis o] Kinetic Data In the detection of structural changes through the rate of proteolysis, qualitative comparison of the initial rates is frequently sufficient to indicate whether there is exposure and its approximate extent. In contrast, a quantitative assessment of the number of exposed bonds susceptible to cleavage requires a protein substrate which does not change conformation (the number of exposed bonds) over an extent of proteolysis sufficiently large to permit analysis of the initial stage of the reaction according to some standard kinetic treatment. The preceding discussion has indicated that this behavior can be expected only when relatively stable derivatives are formed. Harrington and co-workers7,s have investigated systems of this sort, the hydrolysis of myosin by trypsin and of collagen by collagenase. In these cases fast and slow reactions had sufficiently different rate constants that their kinetics could be studied independently. The fast reaction, which produced the meromyosins from myosin, was shown to have little effect on the folding of the molecule, and, in contrast, the subsequent slow reaction, in which 4 times as many bonds were cleaved, led to a significant conformational change. Location o] Bonds Hydrolyzed The study of derivatives of high molecular weight has been discussed, in preceding sections, with reference to defining exposed regions of the polypeptide chain. Although the techniques employed must be developed and will be different for each particular protein, certain comments are appropriate. Separation methods for protein mixtures have reached a high state of development, and although isolation of one reaction product from a complex mixture is difficult it is no longer an overwhelming problem21 The location of sites of cleavage is most fruitful for proteins the sequence of which is known or under study, and in such instances relatively simple analyses of the purified derivatives may be sufficient. For example, in the tryptic hydrolysis of thermally unfolded ribonuelease, cleavage at Lys-Ser (31-32) and Arg-Asp(NH2) (33-34) was established by using only amino acid and end-group analyses. 16 In more complex cases, methods of the sort employed in the original sequence determination become necessary23 *1See Vol. I [I1, 12, 13] and Vol. IV [1]; see also footnote 32. H. A. Sober, R. W. Hartley, Jr., W. R. Carroll, and E. A. Peterson, in "The Proteins" (H. Neurath, ed.), 2nd ed., Vol. III, p. 1. Academic Press, New York, 1965. u See the appropriate sections of this volume, and also footnote 34. R. E. Canfield and C. B. Anfinsen, in "The Proteins" (H. Neurath, ed.), 2nd ed., Vol. I, p. 311. Academic Press, New York, 1963.
[91]
IMMUNOLOGICAL
TECHNIQUES
917
Relationship to Other Methods ]or Investigation o] Con]ormational Changes The kinetic basis of this procedure differentiates it from equilibrium measurements such as titration or chromophore perturbation. In contrast, however, there is a strong similarity to hydrogen exchange,35,se which like susceptibility to proteolysis can reflect flexibility (motility) of the protein. Specifically, a partially unfolded or expanded state may be improbable and of no significance for equilibrium behavior, but so reactive that it dominates kinetic processes such as proteolysis or exchange (in Fig. 2, KNo.0 might be small, but ko,1 if sufficiently large could still dominate kl, o). A conformational change detected by an altered rate of proteolytic cleavage may represent (although not necessarily) an increase in the fraction of molecules in an expanded conformation, this fraction even after its increase being so small as to be unimportant for the equilibrium properties. An effect of this sort should be distinguished from one in which the average conformation is disorganized to a significant extent. It is of interest that the sensitivity of ribonuclease to chymotrypsin develops at temperatures in the lower part of the range for the transition determined by optical methods. 16,~9 Also, although a peptide bond of a native protein may be inaccessible in the most probable or in the average molecular conformation, in spite of this proteolysis could see the bond as "exposed" provided it had a high probability of exposure relative to other bonds. Questions of this sort should be resolved after data have been accumulated for crystallographically defined proteins, but until then, the foregoing broader understanding of protein conformation and conformational changes must be kept in mind when interpreting proteolytic reactions. See this volume [85]. A. Hvidt and S. O. Nielsen, Advan. Protein Chem. 21, 287 (1966).
[91] I m m u n o l o g i c a l T e c h n i q u e s ( G e n e r a l ) B y RAY K. BROWN
This chapter concerns: the preparation of antisera; general tests for identification of the immune system and of impurities; and the precipitin reaction. Careful studies require a variety of techniques. Complement fixation, a micro form of which is described in the following chapter, hemagglutination, and fluorescence are other important techniques. The inhibition of these reactions is often studied. Radioactive tracers are also useful, especially for systems with nonprecipitating antibody.