Preliminary investigation of the behavior of lysozyme in urea solutions

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

66, 182-193 (1956)

Investigation of the Behavior of Lysozyme in Urea Solutions

Preliminary

J. LQonis From the Hormone

Research Laboratory,

California, Berkeley,

University

of

California

Received June 11, 1956

How far protein enzymes can retain their catalytic activity when subjected to conditions likely to bring about denaturation is a problem in which lately there has been a considerable renewal of interest. Recent investigations in the Carlsberg Laboratory have shown that extensive unfolding of the molecule does not destroy the enzymatic activity of ribonuclease (l), whereas the same treatment results in a fully reversible inactivation of trypsin or in an irreversible inactivation of cw-chymotrypsin (2). In the present communication will be reported a similar investigation with lysozyme, which also belongs to the group of relatively low molecular-weight enzymes. This enzyme is further characterized by a rather high cystine content, slightly higher indeed than that of ribonuclease. The possibility that such strong intrachain bonding might be responsible for the well-known heat stability of lysozyme seemed to call for an investigation of the integrity of the tertiary and secondary structures of the molecule under different conditions of denaturation. This problem is of particular interest since, in a very recent study by Josefsson and Edman (3), it was demonstrated that even the primary structure of this enzyme can be reversibly altered, with a concomitant reversible loss of activity, during the N-acyl --f 0-acyl migration. MATERIALS

AND METHODS

Lysozyme A sample of crystallized egg white lysozyme from the Armour Laboratories (lot 003Ll) was used in all experiments; this preparation was a gift obtained through the courtesy of Dr. W. F. White. Drying overnight in vacua at 105” showed 182

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the material to contain 6.1 f 0.07% moisture. The nitrogen content was 18.4~ f 0.1% on a dry-weight, ash-free, basis (expected: 18.6); the ultraviolet absorption spectrum agreed within O.2-O.4o/0with published data.

Reagent-grade urea, from the Merck Laboratories, was used without recrystallization. The sample employed presumably contained a small amount of ammonium carbonate, since the addition of trace acid was required to bring aqueous solutions to pH 7.0.

Microorganisms1 megatherium, strain KM, was grown in Difco peptone and harvested according to directions given by Weibull (4). After preliminary washings with phosphate buffer (0.03 &f, pH 7.0), the cells were treated with phenol (l’% in water; 1 hr. at room temperature), washed extensively with water, and finally lyophilized and stored dry under refrigeration. The yield of dry cells was approximately 2 g. from cultures amounting to 3 1. Bacillus

Micrococcus

was purchased from Difco Laboratories, in the form ultraviolet-killed dry material (Bacto-Lysozyme Substrate).

Zysodeikticus

of a standardized

Viscosity The viscosity of lysozyme solutions was measured by means of an Ostwaldtype capillary viscometer, in a thermostat at 25.8 Z!Z0.03”. A minimum of 1 ml. of solution was required for measurement; the capillary, with several bends, had a total length of approximately 70 cm., and a rather wide bore (co. 1 mm.). Under these conditions, flow times were of the order of 80 sec. for aqueous phosphate buffers; they were measured to the nearest 0.1 sec. No attempt was made to correct flow times for the kinetic-energy term, which in viscometers of this type is low; however, flow times were always corrected to the same driving head of liquid, corresponding to a volume of 2.00 ml. of solution in the particular viscometer employed. The viscosfty of lysozyme in each urea solution was measured at five different concentrations of the protein, in the range between 0.5 and 3%. As a first approximation (see discussion below), the relative viscosity of a protein solution was taken as the ratio between flow times for the solution and for the solvent, respectively; intrinsic viscosities were then computed in the usual fashion from relative viscosities.

Optical Rotation The optical rotation of lysozyme solutions was determined with a Bellingham and Stanley polarimeter, at the temperature of the dark room (24 f 1”). Two tubes were used, measuring 2 dm. and 1 dm. in length, and requiring volumes of cu. 7 ml. and cu. 2 ml. respectively; protein solutions were made at concentrations between 1 It is a pleasure to acknowledge the friendly assistance of Dr. J. De Ley in connection with many problems related to microorganisms.

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1 and 3oJ,in order to achieve a reading difference of 1.3-1.7’ between solution and solvent. Twenty readings were made for each solution, with each reading taken to the nearest O.Ol”, and averaged. The standard error was ~f~O.3-0.4”in terms of specific rotation, and became of the order of ~0.5” when errors in weighing of the sample were allowed for.

Lytic Activity (a) “Turbidimetry.” To determine the lytic activity, a procedure similar to that of Boasson, although slightly modified, was employed with both microorganisms (5). The microorganism in concentrated suspension is diluted with the solvent (water or urea solutions, all containing 0.03 M phosphate buffer) to give an optical density (O.D.) of cu. 0.5 at 500 mp (read in a Beckman DU spectrophotometer, withl-cm. cuvettes) ; this O.D. corresponds to cu. 0.6 mg. of dry bacterial cells/ml. It should be mentioned that the sample of M. Zysodeikticus from Difco gave coarser and less stable suspensions than the B. megatherium prepared according to Weibull. To 5 ml. of the buffered suspension (pH 7.0 for B. megatherium; pH 6.2 for M. Zysodeikticus) are added cu. 100 pl. of lysoeyme (N-50 pg. of enzyme for the former; 2-10 pg. for the latter), and the reaction mixture is allowed to react at room temperature with frequent shaking. Optical-density readings at 500 mp are taken every fifth minute in succession or at a predetermined time interval, generally 20 or 30 min. The change in O.D. is relative to a blank containing bacterial cells but no lysozyme. (b) “Permeability.” Since the above method obviously measures a very complicated process, it seemed advisable to confirm the results by means of a different technique. Accordingly, the liberation of cell constituents through damaged cell membranes was estimated by following their appearance in the suspension solvent. The first part of the experimental procedure is identical to that followed in the “turbidity” method. At a predetermined time (15-30 min.) the reaction mixture is centrifuged to remove intact cells and cell fragments (5 min., 3500 r.p.m.). The O.D. of the supernatant fluid is next read at 260 rnp for nucleotides and nucleic acids, and at 280 rnp for proteins; undiluted supernatant fluid could be read directly in the Beckman spectrophotometer, model DU, with l-cm. cuvettes, starting with 0.3-0.6 mg. of dry cells/ml. With either method, a reproducibility of about 10% is obtained.

RESULTS AND DISCUSSION

Structural Properties of Lysozyme in Urea Solutions When the relative viscosity of a protein solution is computed directly from the ratio of two flow times in the viscometer, without correction for the slight difference in specific gravity between solvent and solution, a value is obtained which is in fact a kinematic viscosity. By the same token, extrapolation of the linear plot relating reduced viscosity2 to * (~,~a= q..JC = (qrel -1)/C), where C is the protein concentration; calculation, concentrations have been expressed in grams of lysozyme/ml.

for this of solu-

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SOLUTIONS

concentration leads to an intrinsic kinematic viscosity. The latter quantity is plotted in Fig. 1 as a function of the urea concentration in the solvent. It is readily apparent that the intrinsic viscosity of lysozyme, although rather constant in the range between 0 and 5 M urea, increases markedly above this point. 4.0-

35i

c FE r-l ,

3.0-

2

0

-

4

6

8

IO

Urea Concentration (moles / Ibtre)

FIG. 1. Intrinsic kinematic viscosity and specific optical rotation of lysozyme in urea solutions. All solutions were 0.03 M with respect to phosphate buffer, at pH = 7.0. Upper curve and open circles represent viscosity data (left scale) ; lower curve and solid circles represent rotation data (right scale).

If the accepted value of 0.722 for the partial specific volume of the protein [cf. (S)] is used, the intrinsic kinematic viscosity of lysozyme in buffered water solution can be converted into a true intrinsic viscosity [cf. (7a)]. This quantity, (2.5 f 0.1) (g./ml.)-‘, after correction for the slope of the plot of Qed vs. C, and for electroviscous effects [cf. (7b)], is in close agreement with the value for r]redreported by Jirgensons, for 1% lysozyme in salt-free solution (8). However, our data seem to be at variance with the value of approximately 5(g./ml.)-1 reported in graphical form by Yang and Foster (9) ; there is no obvious explanation for this discrepancy. The value for intrinsic viscosity may be used to gain some idea of the tion. The slope of the plot of PJ,~versus C is (0.30 f 0.04) X lO+z in the range between 0 and 7 M urea, and increases to (0.60 f 0.06) X lO+z at the 10 M urea level.

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hydrodynamic structure of the molecule; as a first approach, the equation of Simha [cf. (6)] can be utilized for this purpose. Assuming the protein to be unhydrated in 0.03 M phosphate buffer, it is then found that the lysoayme molecule approximates a prolate ellipsoid with an axial ratio of 3: 1. A more involved treatment of the data, as outlined by Sadron (10) and by Scheraga et al. (ll), yields substantially the same result, indicating that the hydrodynamic ellipsoid equivalent to the lysozyme molecule can be described as a compact, unhydrated particle with an axial ratio not higher than 3 or 4. This seems to be reasonable, not only in view of the high cystine content and of the crystallographic properties of the protein, but also in the light of data derived from x-ray scattering and dielectric-constant studies [cf. (S)], as well as from a consideration of electrostatic effects upon the titration curve (12). However, this approach of Sadron and of Scheraga requires values for two additional parameters: molecular weight and sedimentation constant. For the former, we took the value of 15,000, based on amino acid composition of the protein [cf. (13)], and for the latter, 2.11 X lo-l3 sec. (14). It should be noted that, since various conflicting data have been reported, a value for the sedimentation constant lower by as much as 12 % could have been adopted with equal justification; if such a lower value is taken, the equivalent hydrodynamic ellipsoid approximates more closely a spherical, somewhat hydrated, particle. However, no matter what value is chosen for the calculations, the results still favor the impression that lysozyme in its native state is a protein of very compact and symmetrical structure. The increase in intrinsic viscosity of lysozyme that is observed as urea denaturation proceeds might be indicative of an increase in either molecular asymmetry or hydration, or both simultaneously. Although viscosity data alone are insufficient to indicate which factor is the more important, we favor the hypothesis of an increasing degree of swelling without appreciable change in symmetry. This view seems more compatible with the relatively small concomitant change in the secondary folding (see discussion on optical rotation, below); it is also more in line with the results of recent investigations on the denaturation of other proteins (11, 15). Changes in specific optical rotation of lysozyme are recorded in Fig. 1. Our plateau value of -49.6 f 0.5”, in water or at low concentrations of urea, is in very satisfactory agreement with previous data which range between -46.8’ and -52” (8, 9). A steady increase is observed with concentrations of urea higher than approximately 6 M.

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Our data with 10 M urea do not necessarily represent complete denaturation of the molecule, as evidenced by the fact that the optical rotation curve does not flatten out into a plateau, and by the relatively small variation in optical rotation values which we have observed with urea concentrations between 0 and 10 M (A ‘v 12”). In similar investigations by Jirgensofis (8), where the enzyme was studied in concentrated (5 M) guanidinium chloride at approximately the same temperature, more nearly complete denaturation was presumably achieved. If we accept these latter data as corresponding to the maximal extent of denaturation likely to be obtained, then we can express our values relative to them. Accordingly, denaturation by 10 M urea can be seen to correspond to 90 % of the maximal viscosity change reported by Jirgensons and to only 33 % of the maximal change in optical rotation. In this connection, recent experiments with collagen and gelatin (16) or with synthetic polypeptides (17) have indicated a close correlation between changes in optical rotation and unfolding of the secondary structure of peptide chains, such as cu-helices;these results substantiate previous evidence to this effect (18). Furthermore, if we consider the initial changes in intrinsic viscosity of lysozyme as indicative of alterations occurring mainly in some labile structure of the molecule, the denaturation of the protein can be fitted into a rather simple picture. Outside the range of from 0 to 4 M urea, where apparently the molecule undergoes scarcely any change at all, there is a sharp increase in intrinsic viscosity, which at a concentration of 8 M urea reaches about 60% of the maximum effect (still relative to denaturation in guanidinium chloride) ; on the other hand, at this concentration and under these conditions, the specific rotation has changed by a mere 8 %. At this stage, we may picture the swollen protein molecule as having had the most labile parts of its tertiary structure rather markedly destroyed, whereas the secondary structure is still barely affected. Indeed, the numerous -S-Sbridges, which restrict secondary folding to areas of somewhat limited size, might very well contribute to keeping those areas rather tightly entangled and protected inside the compact particle; accordingly, only a very extensive denaturation of the molecule as a whole would allow the urea to reach some folded configurations of apparently higher stability. In short, the data reported here can be interpreted as * Comparison with other data obtained by Jirgensons at high temperature in the presence of 5 M guanidinium chloride would be less justified, since these latter experiments presumably involved heat denaturation effects superimposed on competition for hydrogen bonds.

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showing a deeply buried secondary folding which is, so to speak, “hidden” or “masked” by some kind of surrounding organization. Lytic Activity of Lysozyme in Urea Solutions

Results obtained with the two methods, “turbidimetry” and “permeability,” that were employed for studying the lysis of either R. megatherium or M. lysodeikticus in the presence of urea, are summarized in Fig. 2. The lytic activity in the presence of urea is expressed as per cent of the lysis observed in the absenceof urea, but under conditions that are identical in every other respect. It should be added that the “permeability” method, which measures the diffusion of cellular materials outside the cells of the microorganism, consistently gave higher activity when the activity was measured at 280 rnp (upper edge of blocks, Fig. 2) than when it is measured at 260 rnp (lower edge of blocks, same figure) ; this obviously reflects a difference in molecular size between polynucleotides and proteins. 2cor

150-

loo-

50-

0-

2

-Urea

4

6

8

I

Concentrohon (moles / Iltre)

FIQ. 2. Lytic activity of lysozyme in urea solutions. All solutions were 0.03 M with respect to phosphate buffer. Upper curve and solid blocks or circles represent lysis of B. megatherium (pH 7.0); lower curve and open blocks or circles represent lysis of M. Zysodeikticus (pH 6.2); circles are for the “turbidimetry” method, blocks for the “permeability” method (see text).

As a general comment with respect to Fig. 2, it should be emphasized that activities measured at low urea concentrations appear higher than those measured in the absence of urea. Microscopic observation of the bacterial cells during and after lysis showed that urea induces a marked

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swelling of killed cells suspended in water [cf. (19)], with a concomitant and nearly instantaneous decrease in the optical density of the cell suspension. It is indeed well known that urea diffuses readily through biological membranes. The swelling presumably enhances the lytic process either by exposing sensitive spots of the cell wall or by favoring adsorption of the enzyme onto the wall; this enhancement is somewhat reminiscent of the synergism observed when lysozyme is allowed to act on microorganisms in the presence of proteolytic enzymes (20). The temperature coefficient of the lytic process was measured in the range of from 1.5 to 4O”C., and found to be absolutely identical whether or not urea (1 M) was present; this undoubtedly means that the lytic action consists, in each instance, in the splitting of the same bonds in the substrate, although those bonds can be more or less easily accessible to the enzyme. Also, in the presence of urea, a shift in the pH optimum of lysozyme, amounting to a few tenths toward the alkaline side, was observed; since this effect was obtained in 2.5 M urea, that is, in a range where the lysozyme molecule is entirely unaffected, it must again be indicative of alterations in the substrate itself. Indeed, killed cells are more sensitive to the action of urea than are the living cells; when tested against young preparations of living B. megatherium, or against aged cultures consisting mainly of living spores, lysozyme manifested activity between 80 and 120 % at a urea concentration ranging from 0 to 5 M. Living cells, however, were not used routinely for activity measurements since they behave in a somewhat erratic manner. In order to test the possibility of a protective action on the part of the substrate against lysozyme denaturation, experiments were performed in which either the substrate or the enzyme is submitted to a preliminary incubation with urea (1 hr. at room temperature; 5 and 10 M urea). No differences in activity could be detected. It is clear from Fig. 2 that B. megatherium and M. lysodeikticus manifest different sensitivities to the action of lysozyme in urea. It is possible that the manipulation of the cells prior to measurement of the activity is partially responsible for this difference in the behavior of the two microorganisms, since a small-scale experiment with phenol-killed M. lysodeikticus gave a still different activity curve, located between those for ultraviolet-killed M. Zysodeikticus and for phenol-killed B. megatherium, with a plateau appearing between 1 and 5 M urea (lysozyme activity N 140 %). Again, the “turbidimetry” and “permeability” methods give different curves with B. megatherium, up to a level of 5

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M urea, whereas the two techniques agree nicely in the case of M. lysodeilcticus. While these marked variations in activity curves show that lysis of whole cells is undoubtedly a very complex process, some common features can be observed between the two microorganisms. Although in the range between 0 and 6 M urea the reaction is obscured by the apparent increase in activity due to a change in the substrate, as denaturation of lysozyme proceeds, both curves point strongly to a vanishingly low lytic activity; the loss of activity roughly parallels the change in intrinsic viscosity discussed above. It seems without doubt that, in contrast to what was observed with ribonuclease (l), even a comparatively mild denaturation of lysozyme is accompanied by the loss of some property necessary for the lysis of sensitive microorganisms.4 It should be added that inactivation by urea represents a typically reversible alteration. When a solution of 1.4 % lysozyme in 10 M urea, after storage during 3 weeks in the cold, was diluted with water to l/1000 and assayed within a range of concentrations against aqueous suspensions of each of the microorganisms, complete recovery of activity was obtained. If anything, there was even a low degree of activation observed (activity equal to 105 % against B. megatherium and 130% against M. lysodeikticus), which cannot be accounted for by the 0.01 M urea present in the assays. GENERAL

CONCLUSIONS

This study may be considered as a preliminary investigation in the sense that we are not entirely satisfied of the homogeneity of the enzyme, and that we further doubt that lytic activity against whole bacterial cells, as used here, is’the best measure of the enzymic function of the protein. With respect to homogeneity, it has been known for some time that crystalline lysozyme does not satisfy the conditions for reversible spreading of the boundary in free electrophoresis (21). The use of refined fractionation techniques has shown more recently that one major and two minor components are present; these can be identified and estimated by either ion-exchange chromatography (22a), counter-current distribution (22b), or electrophoresis on starch (22~). A preliminary experiment 4 Preliminary studies have shown that a lysozyme solution in 4 M guanidinium chloride has no lytic activity whatsoever against B. megatherium; however, this effect may be due to the high ionic strength of the solution, since a high concentration of sodium chloride also causes inactivation [cf. (%)I.

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with ion-exchange chromatography showed that the lysozyme employed in the present investigation contains approximtely 80 % of the major component. As far as lytic activity is concerned, it is quite obvious that the clearing of a cell suspension is a complex process in itself, even without the further interference of the effects of urea upon cell walls. From a rapid survey of the relevant literature, it is apparent that many steps are involved, including adsorption of the enzyme, attack of any capsular material that may be present, disruption of inter-cell membranes, swelling of the wall of isolated cells, and clearing of the bacterial protoplast by autolytic enzymes. Although the use of isolated cell walls (23) or of crude polysaccharide fractions of bacterial origin (24) has been suggested, these methods afford only a limited solution to the many problems involved. Clearly a homogeneous and soluble substrate, preferably a lowmolecular-weight compound of known structure, would be better suited to a more refined kinetic investigation of the reaction (cf. the use of uridine 2’ ,3’-phosphate for ribonuclease). The action of 6-8 M urea upon lysozyme was known to bring about some alteration of the molecule, as evidenced by an increased reactivity of the phenol or -S-Sgroups of the protein (25) or by an increased susceptibility to chymotrypsin (26). On the other hand, there also had been some reports that urea scarcely affects the lytic activity of the protein, an opinion still encountered in a rather recent review (27). A more recent investigation by Dickman and Proctor (28), however, showed that 4 M urea has a very peculiar effect on the lysis of Sarcina lutea by lysozyme: both an activation, as measured by the initial speed, and an inhibition, as shown by the smaller amount of total lysis, were observed simultaneously. From the experiments discussed here, it seems clear that alterations in the molecular structure of the enzyme, due to the action of concentrated urea, are responsible for both the decreased lytic activity and the previously reported increase in reactivity of some chemical groups of the molecule. On the ot,her hand, enhancement of the lytic processby moderately strong urea may presumably be ascribed to changesin the substrate itself. The enzymatic property of lysozyme is rather sensitive to denaturation, since a marked loss of activity occurs before any extensive unfolding of the secondary structure becomesapparent. However, we have observed a complete reversibility of the inactivation by concentrated urea, and other investigators have shown how easy is the recovery even

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from alterations in the primary structure of covalent bonds (3) ; it would thus seem that some very stable internal structure of the molecule might be acting as a guide when the loosened bonds are restored to their native condition. This stable “core” need not necessarily be directly involved with catalytic sites of the molecule. ACKNOWLEDGMENT Wearegrateful to Dr. C. H. Li for his stimulativeinterestin this project. SUMMARY The intrinsic viscosity, optical rotation, and lytic activity of lysozyme have been investigated in urea solutions, within the concentration range of from 0 to 10 M urea. Viscosity data show that the protein molecule is a compact and fairly symmetrical unit in its native state, but that it undergoes swelling at urea concentrations higher than 5-6 M. Optical rotation data point to the presence of a very stable folded configuration in the peptide chain of lysozyme. Unfolding occurs only above 7 M urea, and the extent of unfolding in 10 M urea is less than the corresponding increase in swelling of the molecule. Lytic activity was measured by means of two sensitive microorganisms, B. megatherium and 211. lysodeikticus. Lysis by lysozyme is somewhat obscured by the marked effect of urea upon killed cells. It is clear, however, that concentrated urea inhibits the enzymic properties of the molecule; this inhibition is fully reversible. It is concluded that at least part of the tertiary structure of the molecule is necessary for some step in the course of the lytic process. REFERENCES 1. ANFINSEN, C. B., HARRINGTON, W. F., HVIDT, A., LINDERSTR~~M-LANG,K., OTTESEN, M., AND SCHELLMAN, J. A., Biochim. et Biophys. Ada 17, 141 (1955)1 2. HARRIS, J. I., Nature, 1'77, 471 (1956); Biochem. J. 62, 28P (1956). 3. JOSEFSSON,L., AND EDMAN, P., Acta Chem. Scud 10, 148 (1956). 4. WEIBULL, C., J. Bacterial. 66, 688 (1953). 5. BOASSON, E. H., J. Immunol. 34,281 (1938); cf. SMOLELIS, A. M., AND HARTSELL, S. E., J. Bactetiol. 66, 731 (1949). 6. EDSALL, J. T., in “The Proteins,” (Neurath, H., and Bailey, K., eds.), Vol. I, part B, p. 549. Academic Press Inc., New York (1953). 7. (a) TANFORD, C., J. Phys:Chem. 69,798 (1955). (b) TANFORD, C., AND BUZZELL, J. G., J. Phys. Chem. 60,225 (1956). 8. JIRGENSONS, B., Arch. Biochem. and Biophys. 39, 261 (1962); 41, 333 (1952); 67,376 (1955). 9. YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 77,2374 (1965).

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10. SADRON, C., Progr. Biophys. and Biophys. Chem. 3, 237 (1953). 11. SCHERAGA, H. A., AND MANDELKERN, L., J. Am. Chem. Sot. 76, 179 (1953); cf. SCHERAGA et al., J. Polymer Sci. 14,427 (1954). 12. TANFORD, C., AND WAGNER, M. L., J. Am. Chem. Sot. 76,333l (1954). 13. TRISTRAM, 0. R., in “The Proteins,” (Neurath, H., and Bailey, K., eds.), Vol. IA, p. 181. Acad. Press Inc., New York (1953). 14. WETTER, L. R., AND DEUTGCH, H. F., J. Biol. Chem. 192,237 (1951). 15. DIEU, H., BUZZ. sot. chim. Beiges 66,603 (1956). 16. COHEN, C., Nature 176, 129 (1955). 17. BLOUT, E. R., AND IDELSON, M., J. Am. Chem. Sot. 78,497 (1956); DOTY, P., AND YANG, J. T., ibid. 73,498 (1956). 18. KAUZMANN, W., in “The Mechanism of Enzyme Action” (McElroy, W. D., and Glass, B., eds.), p. 70. The Johns Hopkins Press, Baltimore, 1954. 19. STACEY, M., KENT, P. W., AND NASSAU, E., Biochim. et Biophys. Acta 7, 146 (1951). 20. BECKER, M. E., AND HARTSELL, S. E., Arch. Biochem. and Biophys. 53, 402 (1954). 21. ANDERSON, E. A., AND ALBERTY, R. A., J. Phys. & Colloid Chem. 62, 1345 (1948). 22. (a) TALLAN, H. H., AND STEIN, W. H., J. Biol. Chem. 300,507 (1953). (b) CRAENHA~S, E., AND L~ONIS, J., Bull. sot. chim. Beiges 64,58 (1955). (c) RAACKE, I. D., Arch. Biochem. and Biophys. 62,184 (1956). 23. SALTON, M. R. J., Nature 170, 746 (1952). 24. EPBTEIN, L. A., AND CHAIN, E., Brit. J. Esptl. Pathol. 21,339 (1940); MEYER, K., AND HAHNEL, E., J. Biol. Chem. 163, 723 (1946). 25. FRAENKEL-CONRAT, H., Arch. Biochem. 27, 109 (1950); cf. J. Am. Chem. Sot. 73,625 (1951). 26. AMBROSE, J. A., AND LASKOWSKI, M., Science 116, 358 (1952). 27. FEVOLD, H. L., Advances in Protein Chem. 6,187 (1951). 28. DICKMAN, S. R., AND PROCTOR, C. M., Arch. Biochem. and Biophys. 40, 364 (1952).