Int. J. PeptideProtein Res. 12,1978, 155-163 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author@)

THE DENATURATION O F COVALENTLY INHIBITED SWINE PEPSIN

FAIZAN AHMAD and PETER McPHIE

Laboratory of Biochemistry and Metabolism, National Institutes of Arthritis, Metabolism and Digestive Diseases, National Institutes o f Health, Bethesda, Maryland, U.S.A.

Received 16 January, accepted for publication 6 April 1978

Studies are reported on the denaturation o f freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about p H 6 was found to involve a small expansion o f the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced co- operative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.

Key words: circular dichroism; conformation; denaturation, inactivation, viscosity, pepsin.

It is generally believed that the native structure of a protein is that having the lowest free energy of all kinetically available structures, dictated by its amino acid sequence (Wetlaufer& Ristow, 1973). However, this may not be true for those proteins derived from zymogens by the pro- teolytic removal of several amino acids. These may be very important in directing the folding of the zymogen, after synthesis, to a structure close to that required for the active enzyme. The experimental evidence on this point is not clear. Unfolding of a-chymotrypsin by Gdn.HC1 (Martin, 1964), heat or acid (Lumry & Biltonen, 1969) is readily reversible. On the other hand, its native structure could not be recovered after oxidation of its disulfide bonds, whereas that of its inactive precursor, chymotrypsinogen could (Givol et al., 1975). Similar results were obtained with insulin and pro-insulin (Tanford, 1970). It was suggested that this resulted from Abbreviations: DAG-pepsin, swine pepsin reacted with diazoacetylglycine ethyl ester; Gdn.HC1, guani- dine hydrochloride; CD, circular dichroism.

the introduction of breaks in the polypeptide chain during activation, to give multichain proteins.

Swine pepsin is derived from its zymogen, pepsinogen, by the removal of 44 amino acids from its amino terminus, to give a single chain enzyme. The denaturation of pepsin has been the subject of many extensive studies (Bovey & Yanari, 1960). These all showed the reaction to be almost completely irreversible even when disulfide bonds remained intact. In contrast, unfolding of pepsinogen shows complete rever- sibility if the correct precautions are taken (Ahmad & McPhie, 1978). From this, one might conclude that swine pepsin falls into that class of proteins whose active conform- ation is determined to a large degree by the preexisting conformation of its precursor (Tanford, 1968). However, there are also a large number of other acid proteases, mainly of fungal origin, which are not synthesized as zymogens. Since these show extensive struc- tural homology with swine pepsin (Subra-

155

F. AHMAD and P. McPHIE

manian et al., 1977; Hsu et al., 1977), it seems likely that this protein should be capable of regaining its active form. We have designed experiments specifically to test this hypothesis.

To the best of our knowledge, all previous published work on this problem was performed using pepsin produced by the procedure of Northrop (1930). Not only do these prepar- ations contain up to 20% of dialyzable “split products” (Edelhoch, 1957), but the active fraction is heterogeneous. Rajagopalan et al. (1 966) have presented convincing evidence for cleavages at various points along the peptide chain produced by autodigestion during the preparation. Clearly, such breaks could have marked effects on the stability of the protein. We believed that these effects could be elimin- ated by studies on the behaviour of homo- geneous, intact pepsin prepared as described by these authors. Indeed, Dopheide (unpub- lished observations, quoted in Rajogapalan et al. (1966)) found the rate of alkali denatur- ation of freshly prepared pepsin to differ from that of commercial pepsin. However, our initial experiments showed that autodigestion was a major problem also during denaturation of this protein. Consequently, we chose to study the properties of freshly prepared pepsin which was immediately inactivated by reaction with diazoacetylglycine ethyl ester (DAG- pepsin) as described by Lundblad & Stein (1 969). We chose this reagent both for its speed and specificity of reaction and because we be- lieved its small size would have a minimum per- turbing effect on the physical properties of the protein. The results of these studies are des- cribed below.

EXPERIMENTAL PROCEDURES

Material Swine pepsinogen (Lot FG 368810) obtained from Worthington Biochemical Cop., UltraPure samples of Gdn.HC1 purchased from Schwarz/ Mann and glycylglycine ethyl ester hydro- chloride (Lot 35C-5056) from Sigma were used without further purification. Sephadex was ob- tained from Pharmacia Fine Chemicals. All other chemicals were analytical grade.

156

Methods

Preparation of pepsin. Pepsin was prepared by activation of pepsinogen in the manner des- cribed by Rajagopalan et al. (1966). The effluent fractions from the SP-Sephadex C-25 column, which contained pepsin, were pooled and brought to pH 5 by addition of 2 N sodium acetate. Solutions were promptly frozeh in 5-ml lots, containing 2 mg enzyme/d at - 20’ C.

Preparation of inhibited pepsin. Diazoacetyl- glycine ethyl ester was prepared by the pro- cedure of Riehm & Scheraga (1965). The in- hibited pepsin was prepared according to the method of Lundblad & Stein (1969) at room temperature. At different time intervals, 50 pl aliquots were removed and assayed enzymati- cally against acid-denatured hemoglobin (McPhie, 1975). In order to separate inhibited pepsin from other components, the inhibition reaction mixture was passed through a Sephadex (3-25 column preequilibrated with distilled water.

Measurement of autolysis. Autolysis of both pepsin and inhibited pepsin under various dena- turing conditions was measured by addition of equal volumes of 20% trichloroacetic acid. After immersion in a boiling water bath for 6 min, the protein precipitate was removed by centrifugation, and the amount of non-protein substance was estimated from the optical den- sity at 278 nm of the supernatant.

Determination of protein concentration. Dif- ference spectra between pepsin and inhibited pepsin suggested that there is almost no change in absorption at 278 nm on modification. Avalue of 5.1 x lo4 for €278 (McPhie, 1975) was therefore used to determine the protein con- centration of DAG-pepsin.

Viscosity. Viscosity measurements were made in a Cannon dilution type (size 50) viscometer as described previously (Ahmad & Salahuddin, 1974). The times of fall of solvent (to) and that of solution (t) at different dilutions were measured separately 5-8 times. The reduced

t - to 1 - f i2zpo

toxc Po viscosity, qr, is defined as - + ,

DAG-PEPSIN UNFOLDING

where c is protein concentration, J2 the partial specific volume and p o the solvent density (Tanford, 1955). The intrinsic viscosity, [Q], was obtained by fitting values of qr at various values of c to the equation: qr = [Q] + k [Q] 2c, where k is the Huggins constant. All viscosity data are in units of ml/g (Tanford, 1961). Solutions for viscosity measurements were routinely fdtered through millipore filters HA (pore size 0.45 micron).

Difference spectral m easurem en ts Difference spectral measurements were made in a Cary 15 spectrophotometer. For pH dena- turation studies, pH values of protein solutions were adjusted by adding sodium hydroxide and difference spectra were recorded against a pro- tein solution at pH 4.5. All solutions contained 0.15 MKCl and no buffer. On the other hand, for Gdn.HC1 denaturation experiments, known amounts of the protein stock, buffer and 7 M Gdn.HC1 solution, all at the same pH, were mixed in a 2-ml volumetric flask and incu- bated over night. In this case, difference spectra were obtained using tandem cells, where the reference was always pepsin in sodium acetate buffer, pH 4.5, ionic strength 0.15 M. In order to check the reversibility of these transitions, protein was first exposed to extreme conditions to bring about a complete change and then either concentrated hydrochloric acid was added to denatured protein at pH 7, or buffer was added to Gdn.HC1-denatured protein to give lower concentrations of the denaturant.

Circular dichroism m easurem en ts Circular dichroism (CD) measurements were made in a Cary 60 automatic recording spectro- polarometer with a Model 6001 CD attach- ment, at 25” C. The path length of cell used was 0.1 cm. From the observed value of ellipticity, the mean residue ellipticity, [el, at each wave- . .

e A x 10 length was calculated from: [elA =

CXl M O

X - where 0 is the observed ellipticity in 100 millidegrees, Mo the mean residue weight of pepsin (Mo = 107), c the protein concentration (mglml) and 1 the pathlength (cm). The con- centration used in these experiments was in the range 0.0 1 -0.04 g/ 100 ml.

Fractions of a helix, &structure, and un- ordered structure were estimated by “MLAB”, a curve-fitting system programmed for the PDP-10 computer (Shrager, 1970). The refer- ence curves used were the data for poly-L- lysine as described by White (1976).

pH Measurements pH values of solutions were recorded with a radiometer pH meter, 26 using a GK-2321-C combined electrode.

RESULTS

Full advantage of physical techniques to assess the stability of native pepsin could not be taken with commercial pepsin preparations, since solutions of the native enzyme could not be ob- tained free of autolysed products (Edelhoch, 1957). It is also well known that denatured pepsin is a very good substrate for the native enzyme (Northrop, 1930). Thus, all denatur- ation experiments done so far with commercial pepsin indicated an increase in the autolysed products which presumably results from the proteolysis of the denatured pepsin by the ac- tive enzyme occurring as fast as the denatured molecules are formed (Edelhoch, 1957). In addition, in preliminary experiments on freshly prepared pepsin, using a method already des- cribed, autolysis was detected during alkali and Gdn.HC1 denaturation of the native enzyme. In order to eliminate the autolytic activity of pepsin, the enzyme was inhibited by diazo- acetylglycine ethyl ester. No trichloroacetic acid soluble products were observed in DAG- pepsin either brought to pH 7 or denatured by Gdn.HC1. This communication describes re- sults obtained from a denaturation study of this inhibited pepsin.

Characterization o f the native state o f DAG- pepsin Fig. 1 (Curve A) shows the intrinsic viscosity results on inhibited pepsin under native con- dition at 25OC. From the reduced viscosity measurements in ammonium acetate buffer, pH 4.5, ionic strength 0.15M, a value of 3.2mllg for [Q] was determined. This value of intrinsic viscosity is in agreement with those reported earlier for commercial pepsin preparations

157

F. AHMAD and P. McPHIE

I-

0 1 2 3

PROTEIN CONCENTRATION x lo3, g/ml

FIGURE 1 Intrinsic viscosity of DAG-pepsin under different experimental conditions at 25" C: (A) ammonium acetate buffer, pH 4.5, ionic strength 0.15; (B) 0.15 M KCl, pH 7; (C) 6M Gdn.HCl, pH 4.5; (D) 6M Gdn.HC1-0.1 M P-mercaptoethanol, pH 4.5.

(Edelhoch, 1957; Blumenfeld er al, 1960; Bull & Breese, 1975). The viscosity increment was calculated from [q] (Tanford & Buzzell, 1956) and was found to be 4.2 which corresponds to an axial ratio (a/b) of 3.5 and a frictional ratio (f/fo) of 1.15. Thus the hydrodynamic behavior of DAG-pepsin as measured by intrinsic viscosity is consistent with that of a compactly folded globular conformation.

The CD spectrum of DAG-pepsin in am- monium acetate buffer of pH 4.5 and ionic strength 0.15M, shown in Fig. 2, is identical with that of native procine pepsin (cf. Perlmann & Kerwar, 1973). The fractions of a-helix, /3- structure, and unordered structure, calculated as described above, were 0, 0.58, and 0.42 res- pectively. These values are comparable with those predicted by the procedure of Chou & Fasman (1974); using the amino acid sequence

0

-8

-12 u 180 200 220 240

WAVELENGTH, nm FIGURE 2 CD spectra of native and denatured DAG-pepsin at 25OC. Solvent compositions for 1, 2 and 3 corres- pond to those for A, B, and C of Fig. 1. 0, best fit points for native protein; A, best fit points for dena- tured DAG-pepsin in 0.15 M KC1 solution, pH 7. The best fit of the experimental data were determined as described (White, 1976).

of porcine pepsin (Tang et a/., 1973); values are 0.09 for a-helix, 0.51 for &structure, and 0.40 for unordered structure.

Characterization of the denatured states of D A G-pepsin

Denatured state at pH 7 . As can be seen in Fig. 1, in 0.15 MKCl solution at pH 7.0 the intrinsic viscosity increased from 3.2 to 7.8ml/g which is comparable with the value for commercial pepsin under similar conditions (Edelhoch, 1957). Thus these results suggest a slight change in the gross conformation of the in- hibited pepsin at pH 7.

The CD spectrum, shown in Fig. 2, also indi- cated that the polypeptide backbone conform-

158

DAG-PEPSIN UNFOLDING

ation of the protein has undergone a change when DAG-pepsin is alkali denatured at pH 7. The denatured state was found to correspond to a conformation containing 10% &-helix, 28% 0-structure, and 62% unordered structure as determined by fitting CD results to poly-L-lysine basis spectra m t e , 1976).

In addition, the difference spectrum of native protein measured against the protein solution of equal concentration at pH 7.0 (not shown) showed fine structure (two troughs near 292 and 287 nm and a shoulder at 281 nm). This indicated protein unfolding and subsequent exposure of tyrosine and tryptophan residues of the protein at pH 7.0.

Denatured state in 6 M Gdn.HC2. The intrinsic viscosities of DAG-pepsin in 6 M Gdn.HC1 and in 6 M Gdn.HC1 containing 0.1 M 0-mercapto- ethanol were determined at pH 4.5 and 25' C, and the respective values were 28.5 and 31.5 ml/g (see Fig. 1). The former is very much higher than the value of 7.5 ml/g measured for commercial pepsin in 6 M Gdn.HC1 (Bull & Breese, 1975). This discrepancy can very well be explained by the observation that pepsin undergoes extensive autolysis in the presence of guanidine hydrochloride (Blumenfeld er al., 1960). Degradation would lead to a smaller value of [q] for uninhibited pepsin. The Huggins constant for DAG-pepsin in 6 M Gdn.HC1 plus 0.1 M 0-mercaptoethanol was determined to be 0.35 which is usually found for linear randomly coiled proteins (Tanford el al., 1967). For DAG-pepsin in 6 M Gdn.HC1 with disulfide bonds reduced, [q] was cal- culated by the empirical equation for linear random coil proteins (Tanford, 1968) to be 33.7ml/g, which is very close to the experi- mental value. Thus these results suggest that DAG-pepsin, with its disulfide bonds broken, behaves as a random coil.

The intrinsic viscosity of unfolded DAG- pepsin in 6 M Gdn.HC1 is increased by 10% by adding 0-mercaptoethanol. As expected, the cross links due to disulfide bonds decreased the viscosity of the protein. From viscosity measurements on pepsinogen under similar conditions it has been observed that the cross links reduced the value of [q] by 13% (Tanford et al., 1967). The fact that two independent

investigations have produced the same con- clusion regarding the constraints imposed by disulfide bonds in zymogen and enzyme in random coil conformations strengthens our conviction that the previous results on pepsin denatured by 6M Gdn.HC1 are in error (Bull & Breese, 1975).

Fig. 2 shows that the CD spectrum of DAG- pepsin in 6 M Gdn.HC1 at pH 4.5 is different from that obtained under native conditions. The circular dichroic spectrum of the denatured state in 6 M Gdn.HC1 resembles that of unfolded proteins under the same conditions (Dearborn & Wetlaufer, 1970).

The difference spectrum of DAG-pepsin in 6 M Gdn.HC1 was found to have the same characteristics as described for the alkali- denatured protein, except that the magnitudes of the troughs are much larger. This suggests increased exposure of tyrosyl and tryptophyl residues on Gdn.HC1 denaturation.

Isothermal denaturation. The pH induced transition of DAG-pepsin from its native state to the alkali denatured state was followed in 0.15 MKCI, 25"C, by measuring Ae at 292nm as a function of increasing concentration of NaOH. Results are depicted in Fig. 3A, where

represents the decrease in absorbance at 292 nm due to denaturation of a molar solution of protein in a cell of 1 cm light path. The tran- sition thus followed, seems to be complete at pH 7.0. It has been observed that protein dena- tured at pH 7.0 could not be full renatured as judged by difference spectral measurements. On the other hand, it has been found that the CD spectrum (in the far ultraviolet region) of DAG- pepsin returned to 4.5 is indistinguishable from that obtained from the native inactive molecule. This seems to suggest that the polypeptide back- bone conformational change on denaturation, is reversible. It should be noted that above 0.5 mg/ml protein concentration, turbidity devel- oped on renaturation. This made viscosity measurements on renatured pepsin impossible.

The effect of the addition of Gdn.HC1 to the native-and alkali-denatured state of DAG-pepsin is shown in Fig. 3B. Due to the insolubility of both Gdn.HC1 and alkali-denatured pepsin up to 4M Gdn.HC1, complete transition curves could not be observed. It seems that a similar

159

F. AHMAD and P. McPHIE

N n

3 a

TABLE 1 Physical parameters of native and denntured DAG-pepsin

A€, cm2 mol-' [B],degcm' Experimental conditions [ Q ] drnol''

ml/g 287nm 292nm 220 nm

Native, pH 4.5,0.15 M KCI

Denatured, pH 7.0,0.15 M

Denatured, pH 4 . 5 , 6 M

Denatured at pH 7 in 6 M

25" C 3.2 0 0 -7200

KCI, 25" C 7.8 - 1 6 0 0 -1100 -5900

Gdn.HCl,25" C 28.5 -7500 -5500 -1900

Gdn.HC1,2S0 c" 28.6 -7500 -5500 -1900

*Gdn.HCI was added to akali-denatured protein.

PH 5 7

0

-500

- 1 000 L Insoluble Region B

2000

W"

a 4000

6000

8000 0 2 4 6

Gdn. HC1.M

FIGURE 3 (A) effect of pH on DAG-pepsin at 25' C. (B) effect of Gdn.HC1 on native protein (0, o), and denatured protein at pH 7, (0 , m).

denatured state is observed in both courses of denaturation, i.e. Gdn.HC1 acts either on native DAG-pepsin or on denatured DAG-pepsin to give rise to the same product. Viscosity and CD measurements also supported this conclusion. Another remarkable thing about the results shown in Fig. 3B is that the properties of the denatured state are similar t o those observed in pepsinogen denaturation (Ahmad & McPhie, 1978). This seems to suggest that all tyrosines and tryptophan residues are exposed in the Gdn.HC1 denatured state.

DISCUSSION

For these measurements to be relevant to the folding of active pepsin, it is necessary to show that covalent inhibition had no effect on the conformation of the enzyme. Results on in- trinsic viscosity showed that the overall con- formation of DAG-pepsin under native con- dition is globular. From a comparison of the intrinsic viscosity of the native inhibited pep- sin with the values reported for commercial pepsin in the native state (Edelhoch, 1957; Blumenfeld ef al., 1960; Bull & Breese, 1975), it may be concluded that covalent inhibition of pepsin does not bring about any change in the gross conformation of the molecule. Furthermore, identical circular dichroic spectra, observed for both freshly prepared and DAG- pepsin, suggest that the covalent attachment of the inhibitor does not produce any perturb- ation of the polypeptide backbone conform- ation of the active enzyme either. Analysis of

160

DAG-PEPSIN UNFOLDING

these spectra suggested that the native state of DAG pepsin corresponds to a compactly folded conformation containing 58% 0-structure and 42% unordered structure. This is in reasonable agreement with the values measured crystal- lographically for the homologous acid proteases. Close similarity between the conformations of free and DAG-pepsin can also be inferred from the observations that pepsin inhibited by simi- lar diazo compounds can still bind other specific inhibitors, such as 1,2ep0xy-3~p-nitrophenoxy- propane (Tang, 1971) and pepstatin A (Aoyagi & Umezawa, 1975).

A summary of the results characterizing the native and denatured states is given in Table 1. It is clear from the changes in physical para- meters that DAG-pepsin undergoes a substantial conformational change at pH 7. The increase in [Q] at pH 7 where the difference spectral change has reached a plateau value (see Fig. A), pre- sumably represents an unfolded state in which 20% of the buried tyrosyl and tryptophanyl residues are exposed. The CD measurements can be interpreted to show that this denatured state contains 10, 28, and 62% of a-helix, 0- structure and disordered structure respectively.

Unfolding in Gdn.HC1 is often more com- plete than that obtained by pH variation. Tanford and his associates have demonstrated the elimination of all non-covalent structure, with the subsequent adoption of a random coil conformation (for a review, see Tanford, 1968, 1970). The value of [T) ] in 6M Gdn.HC1 con- taining 0.1 M 0-mercaptoethanol is in good agreement with that predicted on the basis of this model: by using the equation of Tanford (1968) for a polypeptide chain of 327 residues, the predicted value of [Q] is 33.7ml/g, com- pared with the observed value of 31.5 ml/g. Thus reduced DAG-pepsin is fully unfolded in 6M Gdn.HC1 and the molecule behaves as ran- dom coil (Huggins constant 0.35). The unre- duced protein in 6 M Gdn.HC1 alone must also be almost structureless but constrained by its three disulfide bonds. The measured value of [Q] in 6 M Gdn.HC1 is 10% lower than that in 6 M Gdn.HC1 plus 0.1 M Fmercaptoethanol, indicating that the polypeptide loops main- tained by three disulfide bonds form a small portion of the total number of residues. Tang et al. (1973) showed that these bonds involve

the pair of residues 45-50,206-210 and 250- 283.

The question whether the structure of DAG- pepsin is identical to that of unsubstituted pep- sin under similar denaturing conditions must be considered. From theoretical studies of the in- fluence of side chains on the dimensions of ran- dom coils, it was observed that substituents beyond the &carbon atom of the side chain have very little or no effect on the rotations about the two dihedral angles of the peptide backbone (Ramachandram & Sasisekharan, 1968). The dimensions of random coils, and properties such as intrinsic viscosity, that measure these dimensions, depend directly on the backbone rotational angles (Tanford, 1968). Since the reaction takes place beyond the 7- carbon atom of an aspartyl residue in pepsin, the modification should not affect the dimen- sion of the enzyme in the random coil conform- ation. Hence the intrinsic viscosity, measured for DAG-pepsin, should be the same as that of denatured pepsin in guanidine hydrochloride.

Earlier viscosity measurements on alkali- denatured pepsin were interpreted to show that the protein behaved as a random coil (Edelhoch, 1957). However, all physical measurements given here suggested that inhibited pepsin dena- tured at pH 7, has different properties from those of the protein denatured by Gdn.HC1. The evidence that alkali-denatured pepsin con- tains ordered structure is supported by the ob- servation of another cooperative transition by addition of Gdn.HC1 to the alkali-denatured protein. Thus, these differences in the physical properties for two different denatured states seem to reflect a real difference in the extent of unfolding to which the native structure has been disrupted, and do not simply represent the effects of solvent compositions. The pro- tein seems to be completely disordered under all final conditions.

' m e failure to achieve complete renatur- ation is the most significant experimental fmd- ing. Two kinds of irreversibility of the pH- induced transition of DAG-pepsin were ob- served. At higher protein concentrations (> 0.5 mg/ml) irreversible aggregation was found, and at the lowest concentration (0.1 mg/ml) where no aggregation was apparent, difference spectral measurements indicated

161

F. AHMAD and P. McPHIE

persistent changes in the environment of the chromophores of the re-acidified protein whose polypeptide backbone conformation seemed similar to that of the native protein. On the other hand, slow or rapid removal of the denaturant from solutions of unfolded protein in Gdn.HC1 invariably caused aggre- gation at all protein concentrations. The formation of interpeptide disulfide bonds in denatured proteins has been reported in many apparent irreversible reactions (Tan ford, 1968). But it was found in all instances that the reducing agent, P-mercaptoethanol, had no effect on the reversibility of the DAG-pepsin transitions shown in Fig. 3, indicating that disulfide exchange plays no role in the form- ation of irreversible denatured product. This seemed unlikely, u priori, since all our experi- ments were performed at neutral pH or below. Thus it seems that failure to observe renatur- ation may arise from causes intrinsic to pep- sin. It was shown earlier that at neutral pH, pepsinogen readily refolds from the random ‘coil form (Ahmad & McPhie, 1978). Below pH 6, native pepsinogen becomes unstable and is activated to pepsin. Under the con- ditions used in this study, activation occurs by a unique, intramolecular reaction (Al-Janabi et ul., 1972), which is accompanied by irre- versible conformational changes (McPhie, 1972). The information for these reactions must be contained in the sequence of pepsinogen. Re- turning pepsin to neutral pH results in denatur- ation, rather than in the regain of pepsinogen’s conformation. Our circular dichroism results show that this denatured protein can form secondary structure similar to that found in pepsin, on again lowering the pH (Fig, 2). How- ever the persistence of the ultraviolet difference spectrum under these conditions, indicates the more subtle changes in conformation to be irreversible. These must underlie the precipi- tation which occurs at higher concentrations of protein and in the presence of denaturant.

As stated in the introduction there are other acid proteases, which have considerable struc- tural and sequence homology with swine pepsin (Subramanian et ul., 1977; Hsu et ul., 1977), but are not synthesized as inactive precursors. Nevertheless, these proteins are able to attain the active conformation. This change in be-

162

haviour may result from differences in their sequences from that of swine pepsin or from some unknown factor in the intracellular environment in which they are formed. These questions might be resolved by similar conform- ational studies on one or more of these fungal proteases.

ACKNOWLEDGMENTS

We would like to thank Dr. Fred White for the use of the spectropolarimeter and his help with the analysis of our data, and Dr. Gary Felsenfeld for the loan of his viscometers.

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