A Comparative Study of the Structures of Bovine and

Ovine Pancreatic Ribonucleases

CHRISTIAN B. ANFINSEN, STIG E. G. &VET, JUANITA P. COOKE, AND B~RJE J~NSSON

From the Laboratory of Cellular Physiology and Metabolism, National Heart Institute, National Institutes of Health, Bethesda, Maryland and Kemiska Institutionen, Karolinska Institutet, Stockholm, Sweden

(Received for publication, October 14, 1958)

The sequence of the amino acids in the single, cross-linked polypeptide chain of bovine pancreatic ribonuclease is now suf- ficiently well known to permit a fairly detailed comparison of this protein with ribonucleases isolated from other biological sources. Such a comparison is of particular interest in the case of an en- zyme molecule since variations in structure from species to species may yield valuable information on the location of the site of enzymatic activity. Thus, modifications in sequence which re- sult in changes in charge distribution or which alter the possibil- ities for cross linkage through hydrogen bonds or through the interaction of hydrophobic side chains might suggest the nonin- volvement of certain amino acid residues in the process of catal- ysis.

The comparative study of the structure of an enzyme from dif- ferent species is also of great interest in relation to the under- standing of the chemical basis of speciation and evolution. It is now well known that a number of biologically active proteins may be considerably modified by degradative methods without inactivation. The question arises at once as to the biological reasons for the perpetuation of the “unessential” parts of these proteins. If mutations occur which lead to the biosynthesis of an enzyme which is nonfunctional, such mutations might be ex- pected to be lethal ones which would not be perpetuated in the heredity of the species in question. On the other hand, muta- tions leading to changes in those parts of the protein not directly required for catalytic activity or to changes which only par- tially modify the efficiency of catalysis might be retained in the strain. It is attractive to suppose that the summation of such “permissible” changes in a number of proteins might be a major factor in the process of speciation and in evolution in general.

In the present paper we wish to report on a comparison of the amino acid composition of peptides obtained from beef and sheep ribonucleases by the action of trypsin and chymotrypsin.

EXPERIMENTAL

Materials and Methods

Commercial crystalline ribonuclease (Armour, Lot No. 381- 059) was further purified on columns of the cation exchange adsorbent, carboxymethyl cellulose (1,2), as described in the ac- companying paper (3). The major peak (commonly termed ri- bonuclease A (4)) obtained from the columns was compared, in the following sequence studies, with “Peak III” of sheep ribo- nuclease. As described in the preceding paper (3), both protein preparations contained the same NHz-terminal amino acid, ly-

sine, and exhibited essentially the same ~20,~ values upon ultra- centrifugation. Their specific enzyme activities were identical.

Duplicate samples of native beef and sheep ribonucleases were hydrolyzed and subjected to preliminary amino acid analyses1 by the method of Moore and Stein (5). Since no study was made of the losses of certain amino acids during hydrolysis (6), and since many amino acids occur in large amounts in ribonuclease, the results were not sufficiently accurate to permit a decisive ap- praisal of the differences observed, and the detailed data are not included here. However, the differences observed between the two proteins were qualitatively consistent with the results of the sequence studies and indicated the presence of less lysine, thre- onine, and aspartic acid, and more glutamic acid and serine in the ovine protein.

For the sequence studies protein samples were oxidized with performic acid by the usual method (7,8). Preliminary digestion experiments with trypsin and chymotrypsin were carried out at 37” in the pH stat (9) at pH 8.0 to determine the time required for completion of proteolytic cleavage of the oxidized chains of the two ribonucleases. Uptake of alkali was complete in less than 2 hours when trypsin was added in an amount equal to 1 per cent of the oxidized ribonuclease, and a second a-hour period sufficed for complete digestion with chymotrypsin, which was added directly to the trypsin digest in the reaction vessel of the pH stat. Fractionation of several such digests by techniques to be described below indicated that the results of the combined digestion with trypsin and chymotrypsin were completely re- producible and yielded identical peptide patterns upon paper chromatography and electrophoresis.

In most of the experiments to be reported, digestions were carried out more conveniently in ammonium carbonate buffer. The peptide patterns from such digestions were the same as those obtained by the pH stat procedure. In a typical experiment 20 mg. of oxidized ribonuclease were dissolved in 4 ml. of 0.1 M

(NHJzC03 buffer, pH 8.0, and 5 ~1. of phenol red indicator were added to permit a visual check on possible changes in acidity during digestion. Trypsin (0.2 mg. in 0.1 ml. of water) was added, and the digestion was allowed to proceed for 2 hours in a water bath at 37”. Chymotrypsin (0.2 mg. in 0.1 ml. of water) was then added, and after 2 hours the entire digestion mixture was lyophilized. The buffer salt was removed by allowing the dried material to stand in a vacuum over PZOS and NaOH pel-

1 The authors are grateful to Dr. Karl Piez of the National Institute for Arthritis and Metabolic Diseases, National Insti- tutes of Health, for carrying out these analyses.

1118

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May 1959 Anjinsen, hpist, Cooke, and J&won

lets for 3 days. The desalted mixture of peptides was dissolved in 1 ml. of water, and aliquots were applied to paper for chro- matography and electrophoresis.

For comparative studies of NHz-terminal sequences, samples of dinitrophenylated-oxidized ribonucleases were prepared as pre- viously described (10). The yellow, ethyl acetate-soluble dini- trophenyl peptides produced by the action of pepsin (0.2 mg. of Armour’s crystalline pepsin, 20 mg. of dinitrophenylated-oxidized ribonuclease, pH 1.8,22 hours, 37”) were purified by electropho- resis on Whatman No. 1 filter paper in ~/30 phosphate buffer at pH 7.0 for 20 hours with the use of a potential gradient of about 25 volts per cm. and 12 ma. Peptides separated in this manner were eluted with constant boiling HCl into sealed tubes and hy- drolyzed overnight (105”), and their amino acid compositions and NH&erminal amino acids were determined by the method of Levy (11)) with slight modifications (10).

As an additional check on the nature of the NHz-terminal se- quence of amino acids in the sheep protein, a 20-mg. sample of the enzyme was carbobenzoxylated and treated with trypsin as previously described (12). This procedure causes the cleavage of the ribonuclease chain at positions following the 4 arginine residues in the molecule. The first 10 residues at the NHz-ter- minal end of the chain appear as a single peptide and the subse- quent 23 as another fragment. The decarbobenzoxylated digest

FIG. 1. Separation of peptide components in a digest of oxi- dized bovine pancreatic ribonuclease. The oxidized protein was digested successively with trypsin and chymotrypsin as described in the text. Paper chromatography of an aliquot applied at the origin, to the left of the figure, was carried out in the horizontal direction, followed hy paper electrophoresis in the vertical direc- tion (cathode at the top).

FIG. 2. Separation of peptides from an enzymatic digest of oxi- dized sheep pancreatic ribonuclease. The conditions were as described in the text and in the legend to Fig. 1.

was fractionated as described below and the two peptides above were eluted, and analyzed, after acid hydrolysis, for amino acid composition by two-dimensional paper chromatography.

RESULTS

Typical fractionations of the enzymatic digests obtained with trypsin and chymotrypsin are shown in Figs. 1 and 2. These were obtained by paper chromatography of 50-111. aliquots of the salt- free digests (containing about 1 mg. of peptide material) on Whatman No. 3 filter paper sheets, followed by paper electro- phoresis for approximately 1 hour at 40 volts per cm. at pH 6.5 in pyridine-acetic acid buffer (13-15). The solvent for chro- matography was n-butanol-H20-acetic acid, 4 : 5 : 1. The aliquots were applied in a small spot (Figs. 1 and 2), and descending chromatography was allowed to continue until the yellow spct of phenol red (present during digestion) had traveled about two thirds of the way to the end of the sheet (approximately !G hours). The sheet was then dried in air and cut into halves, for convenience in handling, in a direction perpendicular to t,he di- rection of chromatography. Each half was liberally moistened with pyridine-acetic acid buffer (pyridine-glacial acetic acid-H20, 5:0.2 :95) with care being taken to allow the buffer slowly to wash the peptides on the paper into a sharp line connecting the chromatographic origin with the phenol red spot. Care at this stage of the operation insures that the peptide pattern, after electrophoresis, will exhibit well defined spots. The use of chromatography before electrophoresis, rather than the reverse procedure as used by Ingram in his studies on hemoglobin (16), appears to be of great value in the production of sharp patterns.

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1120 Comparison of Bovine and Ovine Pancreatic Ribonucleases Vol. 234, No. 5

TABLE I Analyses of Peptides obtained by paper chromatography and

electrophoresis from trypsin and chymotrypsin digestions of pancreatic beef and sheep oxidized ribonuclease*

The spots labeled 5, 17 and 19 in the fractionation of digests of sheep and beef ribonucleases were present in such small quanti- ties that their amino acid compositions could not be determined with any assurance. The same was true for the component la- beled X in the beef ribonuclease patterns.

The intensity of staining of each amino acid on two-dimen- sional paper chromatograms of hydrolysates of the various pep- tides is indicated in a qualitative way by underlining. The sub- scripts, when present, indicate the number of moles of each amino acid in the peptide under consideration, as determined for beef ribonuclease 110. 17).

Beef No.

3b !- 3

@I’

1 16 1 2

13

LysZ, Glu, Thr, Alai (beef) Lys, Glu, Ser, A& (sheep) Phe, Glu, Arg Asp, Glu, Thr, Sera, Met, His Asp, Alan, Sers, Tyr Cys, Asp, Glu, Metz, Lys - Ser, Arg

a and 3b

6t

i 16

1 Asp, Leu, Thr, Lys ASP, Ax

9 18

14 20

7

12 21 15

w

3c

Asp, Leu, Thr, Glu, Arg (sheep) G, Asp, Val, Pro, Thr, Lys, Phe EJ~, VA,, Leu, Serz, His, Asp A&, Cys Asp, Cys, Ala, Vz Ly; Cys, Asp,, Glu, Gly, Thr, Tyr Glu, ser, Tyr Thr, Ser, Met Cys, Asp, Ileu, Thr, Ser, Arg Glu, Gly, Serz, Thr, Lys - Cys, Asp, Ala, Tyrz, Pro, Lys - Asp, Glu, Ala, Thrz, Lys Lys, Glu, Ala, Thr - His, Val, Ileuz,G, Asp, Glu, Gly, Ala, Pro,

Tyr -

10

9 18 14 20

7 12 21

15

w

28

23

28

4 Val,, Pro, His, Phe 4

22 Asp, Ala, Ser, Val 22

\ , ,

Composition Sheep No

From carbobenzoxy-oxidized sheep ribonucleases

Lz, Ser, Glu, Ala, Phe, Arg Cys, Lys, His, Asp, Glu, ST, Met, Thr, A&,

‘I%, Arg

* Amide nitrogens cannot be assigned to specific glut,amic acid or aspartic acid residues on the basis of the present studies since these are removed through cleavage by acid hydrolysis before chromatography.

t According to earlier observations (10, 17), cleavages should have occurred, under the conditions of the experiments reported here, between phenylalanine and glutamic acid in peptide 6 and in such a way as to remove the lysine residue from peptide 8. However, free phenylalanine was not observed and only small amounts of free lysine were detected on the patterns.

$ These two peptides were not visualized on Figs. 1 and 2 by the ninhydrin-staining reaction. However, these parts of the polypeptide chain have been accounted for in peptide Sl as de- scribed in the text.

8 See text.

Further details of the technique employed here will be described elsewhere.2

It is important at this point to emphasize some of the difficul- tics which beset not only those techniques depending on the use of paper chromatography and electrophoresis but on column chromatography as well. These difficulties arise from the inherent contamination of commercially available proteolytic enzymes, even after additional purification, with traces of protease activity different from that characteristic of the major component of the material. Such contamination has been demonstrated fre- quently with a number of protease preparations and may lead to the production of peptide fragments not to be expected on the basis of known specificities for such enzymes as trypsin, chymo- trypsin, or carboxypeptidase.

When an optimal amount of digested protein is taken for pep- tide analysis the patterns are usually clear-cut. With larger loads of material trace components may become visible, some of which may be present in sufficient quantity for analyses. In the present studies we have observed a number of such minor com- ponents when 4 or 5 mg. of digested oxidized ribonuclease were applied to the filter paper sheets and, after chromatography, the papers were run for several hours in the electrophoretic direction to increase the separation of the components. In the case of the ribonucleases studied here these components, with one excep- tion, have shown the same qualitative amino acid composition as a neighboring, more prominent peptide fragment and their prcscncc may be attributable either to deamidation of asparagine or glutamine residues during one of the analytical operations or to some physical reason, perhaps related to adsorption on the paper. The problem is not serious when one deals with a protein of a structure as well known as that of ribonuclease but may become very troublesome with polypeptides with poorly known sequences2 It should also be emphasized that larger peptides frequently react only faintly with the ninhydrin-staining reagent and that, without some prior knowledge of structure such as one has with ribonuclease, considerable portions of the sequence might bc overlooked.2 For the determination of the amino acid compositions of the peptide components shown in Figs. 1 and 2, the papers were first dried at 60-70” until free of the odor of acetic acid and then sprayed with a dilute solution of ninhydrin in absolute ethanol (0.025 per cent). The papers were then dried at 60” until the spots corresponding to those in the above figures were faintly visible. These areas were cut out and the excess ninhydrin reagent washed away by several washings with absolute acetone. The peptides were eluted into small test tubes with constant boiling HCI. The tubes were sealed and allowed to stand at 105” for 16 hours. After removal of the HCl in a vacuum the amino acids were chromatographed, first in butanol-acetic acid-water (see above), and in the second direction, in 80 per cent aqueous pyridine. This two-dimen- sional chromatographic system separates all of the amino acids in oxidized ribonuclease from one another except leucine and isoleucine (methionine sulfone and valine separate well, although methionine runs with valine) and permits unequiv- ocal identification of the peptide in question in relation to the sequence of ribonuclease as presently known.

The composition of the various peptides, together with their positions in the polypeptide chain of ribonuclease, is listed in Table 1. The corresponding peptides in the beef and sheep

Z A. Katz, W. D. Dreyer, and C. B. Anfinsen, unpublished ob- servations.

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May 1959 Anjinsen, &vist, Cooke, and Jhsson 1121

ribonuclease patterns were found to be identical in composition. However, several peptides present in the digest of the beef en- zyme were absent from that of the sheep enzyme and were re- placed by new components. Thus beef peptides 2 and 13 were not found in the patterns obtained from the sheep material, and instead therr was observed the peptide designated 10. This change is attributable to the replacement of the lysine residue in position 37 of the beef ribonuclease chain by glutamic acid in the sheep protein. The action of trypsin produces a trtrapeptide and a diprptidc (designated 2 and 13, respectively) in the former case and a hrxapeptide (designated 10) in the latter.

The region of the pattern from beef digests containing peptides 3a, 3b, and 3c was also different with sheep material. Only a single component could be distinguished in the latter digests, having the composition, (Lys, Glu, Scr, Ala), where the under-

lined residues were present in larger amounts on the basis of the intensity of staining with the ninhydrin spray reagent. Sheep peptides 3a and 3b showed the same composition and it seems likely that 3a is probably due to “tailing” of 3b in the chromato- graphic direction. The amino acid composition above corre- sponds to the first 7 residues of beef ribonuclease, with the thre- onine residue replaced by serine.

Proof of the existence of a species difference in the NHz-ter- minal part of the polyprptide chain was obtained in two addi- tional ways. First, dinitrophenylatcd, oxidized beef and sheep ribonucleases were digested with pepsin as described in “Experi- mental” and in greater detail in a previous publication (10). The NHz-terminal dinitrophenyl-peptides were isolated by paper electrophoresis and hydrolyzed with 6 N HCI for subsequent amino acid analysis by the method of Levy (1 I). Both the sheep and beef proteins yielded a peptide containing 1 mole each of lysine and glutamic acid and slightly more than 1 mole of alanine. The beef peptidc contained, in addition, a residue of threonine, and the peptide from the sheep enzyme contained 1 serine residue. These experiments confirm the existence of a difference in struc- ture between the NH&erminal ends of the chains of the two proteins and indicate that the sequence, Lys. Glu Thr Ala. in beef ribonuclease is replaced by Lys. (Glu,Ser,Ala) in sheep ri- bonuclease III (3). The sequence of the amino acids within the parentheses has not been chcckcd, and inversion of two or more of these residues cannot be ruled out without stepwise degrada- tion of the peptide.

A second confirmation of this species difference was obtained by trypsin digestion of the oxidized sheep enzyme after car- bobenzoxylation. Application of chromatography and electro- phorrsis to such digests, after decarbobcnzoxylation, yielded five major ninhydrin-reactive spots, two of which were eluted and analyzed for amino acid content by paper chromatography (pep- tides S3 and Sl in Table I). Peptide S3 corresponds to the NH&erminal decapcptide sequence of beef ribonuclease, with the replacement of threonine by serine.

The peptide labeled 3a in the patterns from digests of beef ribonuclease cannot be clearly identified on the basis of available information. This peptide has the qualitative composition (Glu, Ala, Lys). The production of a peptide with this composition is not consistent with the action of combined trypsin and chymo- trypsin digestion to be expected on the basis of the amino acid sequence of beef ribonuclease as it is now known. Neither is it to be expected on the basis of the findings of Hirs et al. (17), as presented in their paper on the partial reconstruction of the ribo- nuclease chain from data on partial enzymatic hydrolysis of

oxidized bovine ribonuclease. Further, we have identified a peptide with the same composition in digests which were pre- pared with trypsin alone. Not even traces of a peptide of this composition have been detected in either trypsin or combined trypsin-chymotrypsin digests of sheep ribonuclease III. It seems likely, therefore, that a species difference exists between the bovine and ovine molecules at some position which cannot, at present, be assigned.

A species difference is also inferred by the following set of ob- servations. As shown in Table I, a peptide having the composi- tion (Thrz, Asp (NH,), Glu(NHJ, Ala, Lys) has been isolated and identified by Hirs et al. (17) and has been assigned to residues 99 through 104 in bovine ribonuclease. This peptide was not separated by the usual techniques employed in the present work either with bovine or ovine ribonuclease. However, amino acid analyses of the combined peptide components labeled 3b and 3c were consistent with the presence in this area of this peptide to- gether with 4 or 5 residues from the NHz-terminal end of bovine ribonuclease. The nature of components 3b and 3c was clarified when it was found that aqueous, 80 per cent pyridine, employed as solvent in the chromatographic direction, gave a clean separa- tion of these peptides. Elution and analysis gave the amino acid compositions shown in Table I. No peptide corresponding to 3c was detected in the digests of sheep ribonuclease. However, sheep ribonuclease yielded, instead, peptide 23, the composition of which is very similar to that obtained for beef peptide 3c, ex- cept for the absence of aspartic acid and the presence of a stronger intensity in the spot corresponding to glutamic acid. The findings suggest that the aspartic acid residue at position 101 (present as its amide derivative in bovine ribonuclease) has been replaced by a glutamic acid residue in the sheep protein. The absence of an amide group would have to be invoked to account for the differenre in electrophoretic mobility between peptidc 23 from sheep ribonuclease and peptide 3c from beef ribonuclease Clearly this deduction can be proven correct only by complete sequence analysis of sheep and beef ribonucleases over the regions of the polypeptide chains in question. However, the observations above are internally consistent and suggest a difference between the two proteins in one or more of the amino arid residues between positions 99 and 104.

The peptides which were isolated from the digests of beef ribo- nuclease and of which the amino acid compositions appear in Table 1 account for all of the sequence of the beef enzyme except for the region of the chain from residue 11 through residue 25. According to the results of Hirs et al. (17), this part of the chain should have been represented by two peptidc components whose compositions are given in the table. Their absence from the chromatography-electrophoresis patterns may be attributable to lack of a visible staining reaction with the ninhydrin spray rea- gent. Weak reactivity of certain peptidcs with this reagent is, of course, a fairly common observation and a typical case in point is that of peptide 28, indicated within the broken circles in Figs. 1 and 2.

This region of the chain has, however, been accounted for in the digests of carbobenzoxylated, oxidized ribonuclease, both for the beef and the sheep enzymes, and the two proteins appear to have the same amino acid content within this part of the chain. The amino acids in peptide Sl (in Table I) can have been derived only from that portion of the chain between residues 11 and 33, inclusive, part of which has already been accounted for by pep- tides 16 and 1. Once again, the absence of quantitative differ-

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1122 Comparison of Bovine and Ovine Pancreatic Ribonucleases Vol. 234, No. 5

cnces cannot bc ruled out without cshaustivo sequence compar- ison. However, the color intensities produced by the ninhydrin reaction were qualitatively identical with each of the component amino acids for the two spcc*ics.

DISCUSSION

The studies described above establish that ovine pancreatic ribonucleltsc difiers from the homologous bovincl enzyme by the rcplaclcmcnt of a lysinc by a glutamic residue and of a threonint: by a serint: residue. A third sequence diffcrcncc probably (xxi&s between the two proteins, but is not clearly dcfmablc on the basis of the present data.

The established diffcrenccs arc of interest in relation to the location of the active rcnttr of ribonuclcase within its threc-di- mensional structural. Thcsc species variations are in general ac- caortl with certain predictions which (San bc made on the basis of dcgradative studies. For cxamplr, it has becln found that an enzymatically acative derivative of the cxnzyme can ba prepared by partial reduction of disulfidc linkages (18). The first -S- S- bridge to bc ruptured has bten identified as that joining the 1 st and 6th half-cystinr residues (19). The lysinr-glutamic acid interehangc obsc,rvcd in tht: present study occurs in a portion of the amino acid sequence which lies very near half-cystine residue w I and supports the idea that this may be a catalytically unim-

portant part of the structure of the enzyme. The observed intcr- change introtluces a local change of 2 units of rlcctrical charge. Since ovine and bovine pancreatic ribonuclease exhibit identical specific cnzymc activities it seems likely, cz priori, that a physical modification of such magnitude coultl not have occurred in or near the active cc,nter.

The scrine-threonine interchange at the NHt-terminal end of the polypeptidr chain (residue 3) is a comparatively minor one, since it involves a difference of only a single methylcne group, a situation which has been observed previously in studies on the structural differences among insulins from a variety of species, having identical hormonal activities (20, 21). In the case of ri- bonuck~ase, this interrhangc is of special intc,rest because of the recent work of Richards (22)) who has demonstrated that at least a portion of the NHz-terminal 20 residues of the protein are rc- quired for csatalytic activity or for the stabilization of a critical tertiary structure or for both. The prrsmt findings indicate that residue 3 is either not a part of the rsscntial pcptidr sequence or, alternatively, that the replacement of threoninr by srrine in an essential part of the structure does not introduce significant stere- ochemical difficulties in the binding or hydrolysis of substrate molecules.

The experiments discussed above arc only a few of a consider- able number of studies by many investigators on the species dif-

ferences in proteins and polypeptides (20-24) which begin to form the basis for a rational, chemical approach to the questions of speciation and evolution. If WC assume that the phenotype of an organism is, for the most part, the expression of the properties of its proteins, we must interpret the smallest unit changes in evolution as reflections of modified protein structure leading to alterations in enzymatic or hormonal activities and to changes in the organization of metabolic: and structural systems.

It is obvious that a tremendous amount of structural informa- tion needs to be amassed before it will become possible to make even a preliminary comparison of two species at the protein level. In the mcantimc, since the crossing tcchniqucs of genetics arc ruled out by species incompatibility, a study of the similarities of protein structures as reflections of the genetic information of diffcrcnt species should ultimately prove of great value in estab- lishing the continuity of parts of the gene pool throughout the phyla. Tuppy’s work (25) suggests, for example, that the “gcncs” for caytochrome c in the yeast cell and in the silkworm could bc quite similar to those in the higher vertebrates. If such observations on the basic similarity of homologous proteins throughout the phyla become commonplace, WC shall have fur- ther strong evidence for the existence of a set of essential active centers (26-30) the properties of which have been modified by changes in the “accessory” structure of the protein molecules which contain them.

SUMMARY

Paper chromatography followed by high voltage paper electro- phorcsis has been used for the comparison of digests of oxidized samples of purified ribonucleases isolated from beef and sheep pancreas. All of the significant peptide components separated by this procedure have been subjected to qualitative amino acid analysis by paper chromatography, and the analyses are in ac- cord with earlier studies on the over-all sequence.

Two differcnccs in sequence between the ribonucleases of the two species have been established. One involves the replacement of thrconine at residue 3 in the beef enzyme by serine in the sheep cnzymc and the second, the replacement of a lysine residue at position 37 by glutamic acid. A third difference exists between the two proteins, but its rxact position in the sequence cannot be dcfinittxly assigned without further sequential analysis. The pre- liminary cvidcncc suggests that this difference occurs in the re- gion of the polypcptidc chain between residues 99 and 104.

The rather major change in charge distribution produced by the replacement of lysine by glutamic acid at position 37 is con- sistent with the hypothesis that this region of the native enzyme is not essential as part of the catalytically active center.

1.

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Christian B. Anfinsen, Stig E. G. Åqvist, Juanita P. Cooke and Börje JönssonRibonucleases

A Comparative Study of the Structures of Bovine and Ovine Pancreatic

1959, 234:1118-1123.J. Biol. Chem. 

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