Oct., 1957
STRUCTURAL POSTULATES ON INACTIVATION OF
TRYPSIN MONOLAYERS
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TRYPSIN MONOLAYERS AT THE AIR-WATER INTERFACE. 111. STRUCTURAL POSTULATES ON INACTIVATION BY LEROYG. AUGENSTINE AND B. ROGERRAY Contribution from the Department of Chemktry and Chemical Engineering, University of Illinois, Urbana, Illinois Received November id, 1066
It is postulated that several physical means of inactivating proteins have the following sequential steps in common: (1) rupture of a disulfide bond, (2) breaking of one or more other bonds, and (3) opening of a second disulfide bond which allows the destruction of many intramolecular bonds and leads to a general unfolding of the molecule. Steps 1 and 2 comprise reversible, and if followed by step 3, irreversible, inactivation. The bonds involved in these steps are collectively termed the weak link. It is predicted that an external pressure-a film pressure-can act to maintain molecular configuration, and therefore activity, against both surface and radiant energy effects which would disrupt the molecule. A minimum film pressure should be necessary. Also, the effect of radiation should fall off at higher film pressures. In general, there is agreement between experimental results and prediction.
Introduction Changes in the native properties of proteins (e.g., solubility, optical rotation, viscosity, sulfhydryl titer, aggregation and enzymatic activity) demonstrate what is commonly referred to as denaturation. Such changes are produced by chemical agents (e.g., urea, performic acid, iodinating and chelating reagents, organic solvents, detergents, acids and bases), biological agents (enzymes), as well as by the physical processes of heating, drying, irradiating and exposing at an interface. These changes in many cases represent different kinds and/or degrees of molecular modification and have quite possibly involved dissimilar mechanisms. Therefore, we emphasize that this paper is mainly concerned with denaturation by physical means as measured by the loss in enzymatic activity. Although much effort has been given to determining the kinetics of many types of inactivation, little is known about the exact nature of the mechanism(s) It is particularly perplexing that these very large molecules can, in many cases, be completely inactivated by the production of an ion pair or the absorption of one ultraviolet quantum at a critical site; again, the rate may be increased as much as 100-fold by a 3% increase in temperature.ld3 In general it is assumed that either some critical surface structure is altered or destroyed or else the molecule is partially or completely unfolded.’ For instance, Platzman and Francks have postulated that the production of an ion pair in the molecule causes the instantaneous breakage and reorientation of a large number of hydrogen bonds which leads to inactivation. Doty and Geiduschek6 cite two theories which propose that the primary photochemical effect is the rupture of a peptide bond (1) W. Kauzmann, “The Mechanism of Enzyme Action,” ed. W. McElroy and B. Glass, Johns Hopkins Press, Baltimore, Md., 1954,p. 70-120. (2) H. Neurath, J. Greenstein, F. Putnam and J. Erickson, Chem. Reus., 84, 157 (1944). (3) Our results with trypsin films showed no pronounced temDeratUre effect over several degrees around room temperature. IC should be mentioned that surface and bulk temperatures may possibly be different. (4) A. Mirsky and L. Pauling, Proc. Natl. Acad. Sci., 22, 439 (1936). (5) J. Franck and R. Platzman, “Radiation Biology,” Vol. I , ed. A. Eollaender, McGraw-Hill Book Co., Inc., New York, N. Y., 1954, p. 250-251. (6) P. Doty and P. Geiduschek, “The Proteins,” Vol. IA, ed. H. Neurath and K. Bailey, Academic Press, Inc., New York, N. Y., 1953, D. 393.
as the result of the absorption of an ultraviolet quantum at the site of the bond or at a neighboring aromatic chromophore. Stearn’ has suggested that the rupture of disulfide bonds within the molecule may be responsible and Setlowshas postulated that the ultraviolet quantum yield is related to the cystine content. Barron and co-workerss have proposed the oxidation of sulfhydryl groups on the protein surface. Lumry and Eyring’O picture that “globular proteins consist of polypeptide chains folded on themselves to give hydrogen-bonded secondary structures which are in turn folded in a rigid tertiary arrangement through the interaction of amino acid side chains.” “Reversible thermal denaturation processes are presented as changes in tertiary structure, irreversible processes as changes in secondary structure.” Their model embodies some of the most recent considerations of protein structure and is appealingly simple. However, some data indicate that such a clear delineation between the roles of secondary and tertiary structures may not be valid. For instance, the appearance of the full sulfhydryl titer (tertiary change), either from chemical or thermal treatment is invariably associated with irreversible denaturation. 4,11 Yet treatment with agents that specifically alter secondary structure, such as urea or guanidine, produces reversible denaturation which becomes irreversible upon prolonged standing or further exposure to higher temperatures. lo One of us12 has advanced an hypothesis, summarized below, to explain some of the heat denaturation data of Stearn and others. The individual processes in this scheme are not nem13; rather, the specified sequence of steps leading to inactivation is novel. Hypothesis of Inactivation.-The basic idea is that somewhere in the protein molecule there is a (7) A . Stearn, Aduances in Enzymol., 9,25 (1949). (8) R. Setlow, Biochim. Biophys. Acfa, 16, 444 (1955). (9) G. Barron, S. Dickman, J. Muntz and T. Singer, J . Qen. P k ~ s i o l . , 82, 537 (1949). (IO) R. Lumry and H. Eyring, THISJOURNAL 58, 110 (1954). (11) F. Sanger, Biochem J . , 44, 126 (1949). (12) L. Augenstine, (a) “Information Theory in Biology,” ed. H. Quastler, Univ. of Illinois Press, Urbana, 111.. 1953, p. 119-121; (b) a further discussion of inactivation mechanisms, based upon conditions in multi-dimensional space, is given in “Oak Ridge Symposium on Information Theory in Biology,” ed. H. Yockey, Pergamon Press, New York, N. Y., in press. (13) H.Eyring and A. Stearn, Chem. Revs.,24,252 (1939).
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structural subunit which (a) is readily disrupted, so constituting a “weak link,” and (b) whose integrity is necessary for characteristic biological activity. Three steps are postulated whereby physical denaturing agents produce rupture of the “weak link” and thus inactivation of proteins: 1, opening of a disulfide bond involving a AH of approximately 20 kcal. mole-’ and a small or negligible entropy change’; 2, breaking of one or more neighboring bonds, (e.g., hydrogen bonds), with an appreciable entropy increase; 3, rupture of a second disulfide bond with the formation of a structure that is not reversible to the active state. Steps 1 and 2 produce the activated state of physical denaturation, ie., “mild” or reversible inactivation. The rupture of the second S-S bond, step 3, allows irreversible or so-called “violent” inactivation. Irreversibility could result from the formation of a single new bond.14 However, since it is usually associated with a large increase in entropy, irreversible inactivation probably results most often from the spontaneous destruction of a large number of intramolecular bonds. Thus it would appear that a t least a partial unfolding of the molecule occurs and on this basis the “weak-link” is probably a structure involved in latching the molecule together. Once the second S-S bond has ruptured, the degree of denaturation, Le., amount of change in the native properties, will depend both upon the extent to which unfolding proceeds and upon subsequent reactions of the newly exposed groups of the altered molecule. Energy absorbed from thermal collisions or radiation, or strains imposed upon the molecule due to spreading at an interface, will produce a maximum effect upon the weak link.15 In the absence of sufficient disulfide linkages (for instance, ovalbumin has only a single S S bond,le other easily ruptured bonds which latch the molecule together may be involved in steps 1and 3. However, the data on heat denaturation strongly implicate S-S bonds.4,11,17 The number and the loci of the bonds involved in step 2 are considered to be essentially invariant for a given kind of molecule. On the other hand, an inspection of values of the enthalpy of denaturation a ~ t i v a t i o nwhich ,~ are undoubtedly a function of the number of bonds involved, l1 indicates a wide range of variability between proteins, for example, insulin, 35.6, trypsin, 40.0, and goat hemolysin, 198.0 kcal./mole. Discussion of Experimental Results A main purpose of the experiments in the pres-
ent studiesls is to test the above scheme of inactivation, which is consistent with most of the data on heat denaturation, to the case of inactivation by other physical means. No attempt has been made to determine directly the existence of a weak-link structure; rather, inferential evidence was sought. Here we wish to discuss the data in the light of the hypothesis. The difference between reversible and incipient irreversible inactivation is ascribed to the integrity of the second disulfide bond associated with the weak link (step 3). The role of this intact bond is thus to maintain the molecular configuration so that the other bonds in the weak link which were disrupted (the first S S plus neighboring bonds) can reform to give either the original configuration or a t least one compatible with biological activity. External agents capable of producing a cage effect should, potentially, be able to maintain the molecular configuration even in the event of complete rupture of the weak link; in this manner they would act to protect the molecule. Correspondingly, such agents should produce a decrease in the inactivation resulting from a given treatment. It should be pointed out that McLaren, l9 without considering such specific mechanisms, has also postulated that a Frank-Rabinowitch type cage effect should decrease the level of inactivation. Spreading Effects.-The forces at an interface tending to orient, distort and unfold an adsorbed protein molecule, so as to minimize the free energy, are quite strong and result in maximum numbers of the hydrophilic elements projecting into the aqueous phase and of the hydrophobic elements into the gaseous phase. According to the hypothesis, once the distorting energy exceeds that necessary to completely rupture the weak-link, the molecule unfolds and becomes irreversibly inactivated. However, a sufficient external lateral pressure-the film pressure-by providing a cage effect would counteract this unfolding. Therefore, films formed or allowed to stand at low film pressures should lose their biological activity, whereas, films under high film pressures should retain their activity. A critical minimum pressure should exist below which all activity is lost. Trypsin films at a surface concentration of 10 y / 100 exhibit a film pressure of about 0.25 dyne/ em. (film age of 5 minutes). This appears to be about the minimum pressure for the maintenance of trypsin enzymatic activity since essentially none could be recovered from films of lesser concentration. On the other hand, trypsin spread in excess of 10 y/lOO was recoverable. However, a high film pressure was not completely effective in main(14) Inactivation due to freezing and drying, which apparently is not taining activity since the recoverable activity deaccompanied by a gross opening of the molecule,l1 may depend upon such a process. creased with film age. (This loss possibly may be (15) Platzman has pointed out “that the most stable position for a connected with the known autolytic nature of trypmigrating electron vacancy to become localized” (and thus exert its sin in solution or to non-equilibrium conditions maximum influence) ”18 a t a site that can be crudely identified with existing a t local sites-microscopic surface ripples.} the atom of lowest ionization potential.” See discussion of E. Pollard, “Biochemical Aspects of Basic Mechanisms in Radiobiology,” It was also found that if the film pressure was deNat. Rea. Council No. 307, ed. H.Patt. 1954, pp. 1-29. creased by expansion of the film additional protein (16) G. Tristram, Advances in Protein Chsna., 6 , 83 (1949). unfolded and total activity proportionately de(17) (a) J. Greenstein, J . B i d . Chem., 126, 501 (1938); (b) V. creased. duvigneaud, A. Fitch, E. Petarek and M. Lockwood, ibid., 94, 233 (1931); (c) 0.Wintersteiner, ibid., 102, 472 (1933); (d) K.Stern and A. White, ibid., 117, 95 (1937); (e) G. Miller and K. Anderson, ibid., 144, 4G5 (1942).
(18) (a) R. Ray and L. Augenstine, THIBJOVRNAL; 60, 1193 (1956); (b) L. Augenstine and R. Ray, THISJOURNAL, 61,1380 (1957). (19) A. MoLaren, Advances in Ensymolopy, 9, 76 (1949).
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Oct., 1957
STRUCTURAL POSTULATES ON INACTIVATION OF TRYPSIN MONOLAYERS
Cheesman and Schuller20 have obtained force concentration and force-area plots for pepsin which are somewhat similar to our results with trypsin. Their data are plotted in Fig. 1 as solid lines along with comparable data of ours for trypsin as dashed lines. Their interpretation of the composition of the surface films differs from ours. They propose that only unfolded protein exists in the topmost layer of the surface with native molecules being adsorbed directly beneath this layer. Whenever a native molecule moves into the topmost layer from the underlying solution it unfolds in the process and loses its activity. I n this duplex-film model, the first molecules deposited on the surfaces unfold t.0 cover completely the entire available area, but once sufficient protein has so spread and built up a fairly large film pressure, additional protein collects beneath this topmost layer. According to the findings of Trurnit2I, protein injected into bulk solution beneath a stearate film will not appear in the surface if the pressure of the overriding film is above a certain critical value. As the stearate film is expanded the pressure falls until at the critical value (3-4 dynes/cm.) the pressure suddenly increases again indicating that the force of adsorption of the protein into the surface exceeds the film pressure tending to prevent it. Now if the F-C type film of pepsin were a duplex structure such as this one of stearic acid and protein, then one would expect the plot of Cheesman and Schuller to show a break point, or other discontinuity, such as that indicated by the dotted line in Fig. 1(assume exchsion pressure of 1.5 dyne/cm.) . However, the experimental curves give no evidence of any behavior of this kind. Our interpretation is consistent with the data from both trypsin and pepsin films. Once a sufficient number of molecules have been spread to build up a protective film pressure, additional molecules entering the film do so without unfolding and thus retain their native configuration. That is, globular and unfolded species coexist in the plane of the film in a mosaic-like structure. One estimates that the surface area occupied by a small globular protein molecule will be about '/3 to '/4 that occupied by a molecule completely unfolded and having a thickness of an extended polypeptide chain; therefore, it should require 3 to 4 times as many globular molecules as unfolded molecules to produce equal increments in the film pressure. For instance, pepsin with a molecular weight of about 36,000 gives a ratio of 3.4 :1 assuming a 9 8.thick film. For trypsin one calculates values between 2.8:1 and 3.8:1 depending upon whether a molecular weight of 20,000 or 40,000 is taken and whether one attempts to calculate on an anhydrous or a hydrated basis. Using the data between 1.0 and 3.0 dyne/cm. in Fig. 1, a ratio of molecular increments of 3 . 1 : l is found from Cheesnian and Schuller's data and 4.4 :1 from our data.22 (By using the range above one D.Cheesman and H. Sohuller, J . CoZZ. Sci.. 9 , 113 (1954). (21) (a) H. Trurnit "Monomolecular Layers," ed. H. Sobotka, AAAS, Washington, D.C., 1954, pp. 1-13; (b) personal communications. (22) The 4.4:l is obtained aa follows. In the F-A plot at 1.0 dyne/ om. the film area is 1.35 m.*/mg. or a surface concentration of 7.4 r/100 om.? at 3.0 dyne/cm. the area is 1.00 m.z/mg. or 10 y / l O O cm.2. That is, a change in surface concentration of 2.6 ?/lo0 om.? changed (20)
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large number of resonance structures present in proteins (structures such as N...c:..o~o) 3 and has been demonstrated by Setlow and Doyle30who found that an enzyme which is complexed with its substrate and irradiated in the dry state has a target volume equal to the sum of the molecular volumes. Shore and PardeeZ7have measured the fluorescence efficiencies of dyes bound to proteins, where the initial quantum was absorbed in the protein; and Szent-GyorgyP has presented an extremely interesting discussion of the possible role of water (actually ice) in facilitating energy transport by mediating the formation of triplet states. According to our hypothesis, large film pressures should maintain the molecular configuration, even after the rupture of the second S S bond, for a sufficient time to allow the broken bonds associated (29) L. Pauling, R.Corey and H. Branson, Proc. Natl. Acad. Sci., 87, 205 (1951). (30) R. Betlow and B. Doyle, Rad. Reaearch, 2, 15 (1955).
Vol. 61
with the weak-link to reconstitute in a large percentage of the cases and thim reduce the level of inactivation produced by irradiation. Similarly, very low film pressures should offer little or no maintenance of structure and therefore should not modify the radiation effect. I n agreement with this, irradiation at large surface concentrations and thus large film pressure does seem to produce a lower quantum yield, about 0.01, than the corresponding values, 0.0174.031, reported for irradiations in solution and the dry statemlabRecognizing the limitations of the data, one might say that a high film pressure is "3 effective in reversal of inactivation or else inactivation has a 1:3 chance of proceeding by some as yet unpostulated mechanism. Thus, the hypothesis predicts correctly the direction in which the radiosensitivity changes in films with large surface concentrations; however, it does not account for the change observed in the shape of the dose-response curves at low surface concentrations (see Fig. 4 of ref. 18b).
THE CRYSTAL STRUCTURE OF KCu(CN)z1 BY DONT. CROMER University of Calijornia, Los Alamos Scientific Laboratory, Los Alamos, New Mexko Received March 6, 1967
KCU(UN)~ is monoclinic, space group P2,/c(C;,) with four formula units per unit cell. The cell constants are a = 7.57, The trial structure was deduced from a three-dimensional Patterson followed by a three-dimensional Fourier synthesis. This structure was refined by the least-squares method. All computations were made on the Maniac. The analogous silver and gold com ounds have discrete linear complex ions formed by sp hybridization of the metal. The copper compound has an entirely lifferent structure. The complex ions form spiral polymer chains of composition [Cu CN)2-] The twofold screw axes pass through t,he centers of the chains. The chains are held to ether by the K + ions. $he Cu atom has assumed a threefold hybridized state, probably a distorted sp2 hybrid. The Cu(8N)eunits are held together by a Cu-N bond of 2.05 f 0.02 b. One CN group forms this bond while the other CK group protrudeR from the chain. Good evidence is presented for making a distinction between the C and N atoms. The two Cu-C bond lengths are 1.92 f 0.02 b. The C-N bonds are 1.15 f 0.03 and 1.13 A 0.03 A. The C-Cu-C angle is 134.2 f 0.9".
b = 7.82, c = 7.45 A. and p = 102.2'.
-.
Introduction Compounds of the type KM(CN)z are formed by all of the Group I-B elements. The structure of KAg(CN)Z was determined "by Hoard2 in 1932. In this compound, which crystallizes in the hexagonal system, the complex ion [NCAgCNI- is linear. Although the analogous gold compound, KCU(CN)~,crystallizes in the rhombohedral system, the two structures are very similaraa The hexagonal cell of the gold compound has a c dimension one and one-half times greater than the c dimension of the silver compound while their a dimensions are nearly the same. The gold structure can be derived by properly inserting a layer normal to the c axis in the silver structure. The complex ion [NCAuCNI- is presumably linear also. As part of a general investigation of complex metal cyanides, which is being conducted in this Laboratory (see ref. 4-16), the complete crystal ( 1 ) Work done under the auspices of the Atomic Energy Commission. (2) J. L. Hoard, Z. Krisl., 84, 231 (1933). (3) D. T. Cromer, unpublished work. (4) E. Stariteky and D. I. Walker, Anal. Chem., 18, 419 (1956). (6) E. Staritzky, ibid., 18, 419 (1950). (6) E. Staritzky and F. H. Ellinger, ibid., 88, 420 (1956).
structure of KCu(CN)2 has been determined. This structure has proved to be entirely different from those of the analogous silver and gold compounds. Experimental The crystals of KCu( CN), used in this investigation were prepared by R. A. Penneman, of this Laboratory. The method of preparation of these crystals has been given by Staritzky and Walker4 who also give the space group and cell size. They find that I
