Conformational differences between beef and horse pancreatic ribonucleases

MARZOTTO ET AL.

62

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Biochm.

CONFORMATIOKVAL DIFFERENCES BETWEEN BEEF AND HORSE PANCREATIC RIBONUCLEASES A. MARZO’ITO,

P. PAJETTA,

ANYJL. GALZIGNA

Ccntro Nazionale di Chimica delle Maeromolecole de1 C.N.R. and Istituto di Chimica Organica delI’Universiti di Padova, Padua, Italy

ABSTRACT I. A comparative study was carried out on horse (Equuscaballur) and beef (Bos tam) pancreatic ribonuckase. A different amino-acid composition was found and 5-6 per cent of sugar were detected in purified horse ribonuclease. 2. The coniorznational analysis was performed by circular dichroism, thermal transition, and spectrophotometric titration studies. 3. The difference in catalytic activity of the two enxymes was correlated with their 5tructurc5.

IN recent years extensive studies were carried out by Zen&&n and Barnard (1967) on the homologies of proteins showing the same enzymic function. Such studies were focused on the ixnpor-

tance of changes in primary structure resulting from genetic differences among various vertebrate species and on the effect of the protein structural differences upon the enzymic properties. This work was planned as an investigation in the field of biochemical genetics; it aims to elucidate the importance of conformational changes due to the difference in the aminoacid composition, in order to explain a different catalytic efficiency. Horse pancreatic ribonuclease was recently in isolated, purified, and characterized regard to its enzymic and physicochemical properties by Ishihara, Lie, and Ukita (I 967). In this paper we present data on the conformational differences between the bovine and the horse pancreatic ribonuclease. MATERIALS AND METHODS Bovine pancreatic ribonuclease was obtained from Sigma Chemical Co. and purified as described by Hirs, Stein, and Moore (x953). Horse ribonudcase was extracted and purified from minced pancreas of freshly killed animals

following the salting-out method of Ishihara and othas (1967). The crude enzyme was then purified by chromatography on CG 50 Ambcrlite column (I -8 x go)-*as eluent &2 M aodium phosphate at bH 6.4: desahina was effected on G-25 Sephadcx &lumn cl&d with 5 per cent ace& acid. The final pure fraction of the enxyme was lyophilixed. The ribonuclcase activity was determined according to tither the spectrophotometric method of Kunitz (rg46) or the potentiometric method of Davis and A&n (x955). Roth yeast ribonucleic acid and barium uridine 2’-3’-phosphate were products of the Schwarx Laboratories. The sugar content of the preparation has been checked according to the method of Folin and WI (1920). The removal of su2ars from horse ribonuclease was carried out by &eating the protein with a hydroxylamine solution (Graham, Murphy, and Gottschalk, x963) at pH 8-5 at 25’ C. for 8 hours. After this time the protein was desalted on a (1.8 x go) G-25 Sephadex column eluted with 5 per cent acetic acid and lvo~hiliaed. Then the hydroxamic acid content k&s determined by reaction with ferric chloride. The reaction of Spies and Chambers ( I 949) and the photo-oxidative method (Galiio, Jori, and Scoffone, x968) were employed to test the presence of tryptophan residues. Amino-acid analyses were performed using a Techniwn autoanalyser. Molecular weight was determined according to the gel-filtration method of Whit&a (x963) ; a

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PANCREATIC

c-75 Sepbades column (1-8x 140) equilibrated and eluted with o’ I A4 sodium acetate at pH 5 waa employed. The carboxyl titration measurements of horse

ribonuclease were performed both in o. I 5 M KC1 and in 5 M guanidinehydrochloride with I-2M in urea (G.U.) (Cha and Scheraga, x960). The data were corrected by titration of the solvent alone as a blank. Further details of the individual experiments are given in captions to the iIIustrations. TableI.-Amxm-tam

T

Axamo-AclD

Valine Methionine Isoieucine Leucine Tyl-O&le PhenylaIanine Total

TabL II.-APP.QRENT PK VALUE.5FOR DERlVED

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Horse RibOXlUdSSC

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The amino-acid composition of horse pancreatic ribonuclease is reported in Table I compared with that of bovine protein. The total number of aspartic and glutamic acid residues, after amino-acid analysis, corresponds to 29 groups. Since the titration of horse ribonuclease in G.U. solution has indicated that 12-r 3 carboxy groups are present, the -CONHs content is close to &ZYME

ECLPA2V2~

LysiXlC Histidine Arginine Aspartic acid Threonine Serine Glutamic acid

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123-X27

RESULTS Horse ribonuclease contains 5-6 per cent sugars, even after purification by chromato,graphy at difference with bovine RNase. Repeated hydroxylamine treatments caused the sugar content of the protein to decrease within o’5-o.g per cent, thus showing that the linkage between the protein and the sugar is probably of an ester type, involving the glycosidic hydroxyl group and the carboxyl group of the aspartic and glutamic acid residue. The hydroxamic acid test has proved that about one hydroxamic acid residue is present per mole of the hydroxylamine-treated ribonuclease.

16-17 amid0 groups. Paper electrophoresis and column chromatography measurements support fiuther the larger carboxyl groups content ; horse pancreatic ribonuclease shows, in fact, a higher electrophoretic mobility in 25 per cent acetic acid and a more acidic character on CG 50 Amberlite column eluted with 0.2 A4 phosphate buffer at #H 6.5. .4 molecular weight of about 14,000 was determined for this protein by the gelfiltration method (Whitaker, x963). The pH-activity measurements of hydroxylamine-treated horse ribonuclease, obtained by using o-25 M Tris-HQ buffer, are in good agreement with those reported by Ishihara and others ( I 967). The curve shows an optimal value towards RNA as a substrate at PH 7.3. The optimal activity for bovine measured under the same ribonuclease, experimental conditions, is at PH 6-g. The plc values for both the ribonucleases, calculated according to the procedure described by Dixon and Webb (rg64), are shown in Table II. Fig. I gives the ultra-violet spectra recorded with the Hitachi-Coleman spectrophotometer. The measurements were carried out in aqueous solution at a 6.70 x 10-s molar concentration. Horse R&se, similarly to bovine enzyme, exhibits a peak with a maximum at 277’5 mp, but its molar extinction coefficient, determined as ~=85oo,

64

MARZO’ITO

appears to be slightly smaller than that of bovine ribonuclease. A spectrophotometric titration of the tyrosine residues was then performed after dissolving horse and beef RNase in 0.1 M piperidine brought to different pH values by means of standard solutions of HCl and NaOH. The spectrophotometric titration curves are presented in Fig. 2.

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o-oj M sodium dodecyl sulphate both bands were increased in intensity and the negative band at 207 was shifted to 204 r+. DISCUSSION

AND CONCIJJSION

The amino-acid composition (T&e I) of bovine and horse ribonuclease shows major differences in regard to aspartic acid, glutamic acid, proline, glycine, alanine,

06 -

0.5 O.D. 0.4-

0.3 -

0.2 -

0.1 -

FIG. I.-Ultra-violet spectra of 6.7 x x0-5 M beefj-----) and horse () Rxasc solutions. A Hitachi-Coleman 124 recording spectm&otometer was used. The thermal transition curves representing the structural stability of the two enzymes towards the heat-induced denaturation were also measured; they are shown in Fig. 3. The difference in the conformation of RNase was fkally determined by circular dichroism (C.D.) measurements in the far ultra-violet. The C.D. spectra of horse ribonuclease both in aqueous solution and in the presence of sodium dodecyl sulphate (SDS) are reported in Fig. 4. The C.D. spectrum of horse ribonuclease in aqueous solution shows a minimum at 222 ~rq(l reminiscent of the n +?r+ peptide transition of the a-helix and the dichroic band at 207 + When the enzyme solution was made with

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FIG. 2.-Tyrosine titration curves of beef (-O-O-_) and of horse ribonudeasa (-0-o-j. The molar extinction coefficient EN at 295 xyb is given as a iimction of PH. The &f&rent pH values were obtained using a 0’1 M piperkline buffer adjusted to the desired value by the addition of standard HQ or NaOH. dine, and tyrosine. Such differences suggest the possibiity that the primary structure is not the same in the two proteins. The titration data of home ribonuclease indicate two extra car-boxy1 groups, while the

PANCREATIC

197% 1, 1

RIBO.NUCLEASEs

number of amino groups is roughly the same ; a different ratio in acid-basic residues results with respect to bovine ribonuclease in agreement with electrophoretic and chromatographic findings.

bands is much smaller in horse than in bovine ribonuclease. Furthermore, a different effect is caused by addition of sodium dodecyl sulphate; the greater intensity of both bands and the shift of the negative band from 207 0

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Fro. 3.-Thermal transitions in aqueous solutions of beef (-_O-a-) and hone ribonucleasa (-O-O-) ; [a] at 365 rnp is measured as a function of temperature. The ultra-violet absorption behaviour at 277.5 rnp (Fig. I) reflects the e.xposed aromatic residues and especially the tyrosine residues, since the four phenylalanine residues have a low extinction coefficient. The spectrum is consistent with the tyrosine spectrophotometric titration curve, suggesting that three phenolic residues are exposed and two buried ; in bovine ribonuclease, as is known, three out of six phenolic groups are buried. A comparison of the C.D. spectra shows that the amplitude of the circular dichroic

FIG. e--Far ultra-violet C.D. curves of hone ribonuclease in aqucow soIutions (-) and in ~05 M sodium dodecyl sulphate ( - - - -). C.D. was measured with a Dichrographe II Roussei-Jouan, Paris, at rocm temperature. The sensitivity was kept at I*Io-~. The symbol (0) represents the mean residue molecular ellipticity.

to 204 n+ suggest that the total amounts of or-helix and unordered form are increased. The present data do not permit a decision as to whether the z-helix increase derives from a possible portion of a ,&structure (Tamburro, Scatturin, and Moroder, 1968) or from other regions of the native protein, with the C.D. findings still being different from those of bovine ribonuclease. The differences in C.D. behaviour are in accordance with the previous data of a lower conformational stability for horse ribonuclease, as measured from thermal transition curves, pointing to conformational diversities. Even if this pattern does not permit definitive

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conclusions, it suggests that horse ribonucleate has a less ordered structure than bovine ribonuclease. The conformational difference might finally throw light on the different enzymatic activity and the #I-dependence of the two enzymes. The shift of the pH-activity curve appears analogous to the one observed with partially synthetic ribonucleases (Marzotto, Marchiori, Moroder, 3oni, and Galzigna, I 967)) in which the catalytic efficiency and the pK values of ionizing groups of the reconstituted ribonucleases are affected by modification of the S-peptide structure. REFERENCES CHA, c. Y., and S~HERSOA, I% A. ( r g&f, ‘Structural studies of ribonuclcase. I. Hydrc+ gen ion equilibria in a denaturing solvent’, J.

Am. hem. SOL, 8% 54. F. F., and ALLEN, F. W. (rgsj),

‘The action of ribonuclease on synthetic substrates’,

DA-,

3‘ bid. alem.,2x7, 13.

M., and WEBB, E. C. (x964), &qnms, p. I x8. London: Longmans. FOLIN, O., and WV, H. (rgso), ‘A modified and improved method for determination of sugar’, J. biol. Ckem., 4x, 367. G-20, G., JORI, G., and SCOPPONE,E. (1g68), ‘Selective and quantitative photochemical conversion of the tryptophyl residues to kynurenine in lysozyne’, Biohm. biu~kys. Res. Commun., 31,

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Gw, E. R. B., Muxwsw, W. H., and ‘Studies of mucoGo==--=, A. (wW, proteins’, Biockim. biophy. Acta, 74, 222. Huts, C. W. H., Snuq W. H., and MOORE, S. (x953), ‘A chromatographic investigation of pancreatic ribomaclease’, 3. bid. Ckem., soo, 493. KUMTZ, M. (I g46), ‘ A spectrophotometric method for the measurement of ribonuclease activity’,

Ibid., 164, 563. Isxxa~~11, I_., IR~E, M., and Uxcrr~, T. (rg67),

‘F+sifica~on and properties of horse pancreatic ribonucleases’, J. Biothmr., 62, 430. MARzono, A., MARcnxoxu, F., MORODER, L., Bohn, R., and GALZIGNA, L. (1g67), ‘Enzy-

matic activity of partially synthetic ribonudeases’, Biockim. biopkys. Acta, 147, 26.

SPIES, J. R., and Cwa~s, D. C. (x94$, ‘Chemical determination of tryptophan In proteins’, Anajyt. C&cm.,21, xqg. T--o, A. M., Scxrruw, .4 ., and MORODER, optical rotatory L. (lg68), ‘Far-ultra-violet dispersion and circular dichroism studies of bovine pancreatic ribonuclease A’, Biockim.

biopkys. Acta, 1% 583.

Wxsrr-, mdccular

J. R. (x963), ‘Determination of wc&hts of proteins by gel filtration

on Scphadex’,-Ana&. &m., s, igio ~;ENDZIAN.E. IV.. and BARNARD,E. A. t~g67), _

‘ Diitr&utions ’ of pancreatic ribonucleases, chymotrypsin and trypin in vertebrates’, Arch B&h. Biopkys., 122, 6gg.

iY;y Mror~! I&x: Conformationai analysis, circular dichroism, thermal transition, spectrophotometric titration, horse and beefribonuckase.