the isolation and partial characterization of ribonuclease a from

427Biochem. J. (1973) 135, 427-441Printed in Great Britain

The Isolation and Partial Characterization of Ribonuclease Afrom Bison bison

By GORDON R. STEWART* and KENNETH J. STEVENSONtDepartment of Chemistry, University of Calgary, Calgary T2N 1N4, Alberta, Canada

(Received 20 March 1973)

1. Bison ribonuclease was isolated from pancreas glands of Bison bison by acid extraction,(NH4)2SO4 fractionation, affinity chromatography on Sepharose-5'-(4-aminophenyl-phosphoryl)uridine 2',3'-phosphate and ion-exchange chromatography on Bio-Rex-70.2. The selectivity of the affinity column towards bison ribonuclease in heterogeneous pro-tein solutions was greatly improved by employing piperazine buffers at pH 5.3, whichdecreased non-specific interactions of other proteins. Rapid desorption from the affinitycolumn was obtained with sodium phosphate buffer (pH 3). 3. Bison ribonuclease has atotal amino acid content very similar to ox ribonuclease. Inactivation ofbison ribonucleasewith iodoacetic acid leads to the formation of 0.62 residues of v-carboxymethylhistidineand 0.36 residues of Xr-carboxymethylhistidine. The amino acid composition of peptidesisolated from diagonal peptide 'maps' and also of peptides isolated after pH 1.6 and 2.4two-dimensional high-voltage electrophoresis of a digest of bison ribonuclease labelledwith pyridoxal 5-phosphate indicates that there is complete homology between ox andbison ribonucleases. 4. The Schiff-base attachment site ofpyridoxal 5-phosphate was iden-tified as lysine-41 by NaBH4 reduction followed by peptide isolation.

Barnard (1969) has extensively reviewed variouscomparative aspects and biological function of ribo-nucleases. When comparisons were made amongstribonucleases isolated from pancreas glands of thevertebrates, large variations in amino acid composi-tion and partial amino acid sequence informationindicated that the ox (Smyth et al., 1963), rat andhorse (Beintema & Gruber, 1967) and sheep (Aqvist& Anfinsen, 1959; Anfinsen et al., 1959) had under-gone rapid evolutionary change. The components oftheir ribonuclease active sites, however, were largelyconserved when compared with other segments oftheir structure.With the availability of pancreas glands from the

Plains Buffalo (Bison bison bison), an opportunityexisted to isolate and characterize pancreatic ribo-nuclease from this species of the Bovidae familywhich is very closely related to domestic beef cattle.Such a study was further facilitated by the develop-ments in selective enzyme isolation through affinitychromatography (Cuatrecasas, 1970; Wilchek &Gorecki, 1969).

Experimental

Materials

Sepharose-4B (Agarose; lot 4763) and SephadexG-25 were purchased from Pharmacia, Montreal,

* Present address: Department of Biochemistry,Dalhousie University, Halifax, Nova Scotia, Canada.

t To whom reprint requests should be addressed.

Vol. 135

Canada. NH2pPUP-Sepharose* was prepared in thelaboratory (see under 'Methods') or was purchasedfrom Miles-Yeda Ltd., Rehovoth, Israel (lot 20013).Bovine (ox) ribonuclease A (lot 99B-8020), pyri-doxal 5-phosphate, pepsin and ribonucleic acid (typeVI) were purchased from Sigma Chemical Co.,St. Louis, Mo., U.S.A. Chymotrypsin a was pur-chased from Worthington Biochemical Corp.(Winley-Morris), Montreal, Canada. Porcine trypsinNovo was from the Enzyme Development Corp.,Wall Street, New York, N.Y., U.S.A. lodoacetic acidwas high-purity material from British Drug HousesLtd., Poole, Dorset, U.K. Bio-Rex-70 was purchasedfrom Bio-Rad Laboratories, Richmond, Calif.,

* Abbreviations: RNAase, ribonuclease A (ox or biscnas indicated); NH2pPUP, 5'-(4-aminophenylphosphoryl)-uridine 2',3'-phosphate; NH2pPUP-Sepharose, Sepha-rose to which 5'-(4-aminophenylphosphoryl)uridine 2',3'-phosphate has been covalently attached through the aminogroup; bison RNAase-pyridoxal 5-phosphate, bisonribonuclease to which pyridoxal 5-phosphate has beencovalently attached by NaBH4 reduction of the inter-mediate Schiff base; CmCys, S-carboxymethylcysteine;His(Cm)RNAase, ribonucleaseA (ox or bison) inactivatedwith iodoacetate by modification of histidine-12 andhistidine-119; Hse, homoserine; Hse>, homoserinelactone; Cys(03H), cysteic acid; Met(02), methioninesulphone; Dnp-Lys, N6-2,4-dinitrophenyl-lysine; m.6,the mobility of a peptide or amino acid relative to serineduring high-voltage electrophoresis at pHl.6. Similarnotations denote the mobility of a peptide relative to anamino acid during high-voltage electrophoresis at aparticular pH.

G. R. STEWART AND K. J. STEVENSON

U.S.A. Piperazine was obtained from Fisher Scien-tific Co., Pittsburgh, Pa., U.S.A. CNBr and cyclo-hexyl-3-(2-morpholinoethyl)carbodi-imide metho-p-toluenesulphonate were purchased from PierceChemicals, Rockford, Ill., U.S.A.

Methods

Synthesis of 5'-(4-aminophenylphosphoryl)uridine2',3'-phosphate. The synthesis of NH2pPUP wascarried out essentially as described by Wilchek &Gorecki (1969). Characterization on silica gel t.l.c.by using the detection of phosphate-containing com-pounds as described by Rosenberg (1959), indicatedan RF value of 0.03, compared with the published RFvalue of 0.12 in propan-l-ol-aq. NH3 (sp.gr. 0.88)-water (7:1:2, by vol.). However, the biological speci-ficity of NH2pPUP attached to Sepharose was iden-tical with commercially available NH2pPUP-Sepha-rose.

Coupling ofNH2pPUP to Sepharose-4B. The acti-vation of 100ml of washed, settled Sepharose-4Bwith lOg of CNBr was carried out as suggested byWilchek & Gorecki (1969), Cuatrecasas (1970) andCuatrecasas & Anfinsen (1971). NH2pPUP (210mg,0.35mmol) in 15ml of 0.1 M-NaHCO3 was coupled tothe activated Sepharose in 50% yield as determinedspectrophotometrically at 260nm. The NH2pPUP-Sepharose so formed contained about 1.8,umol ofNH2pPUP/ml of settled Sepharose.

Preparation of2-aminoethanol-Sepharose. WashedSepharose-4B (35ml) was suspended in 70ml ofwater,150u1 (122mg) of 2-aminoethanol was added and thepH was adjusted to 4.7 with HCI. Cyclohexyl-3-(2-morpholinoethyl)carbodi-imide metho-p-toluene-sulphonate (500mg, 1.18mmol) was introduced andthe coupling reaction was allowed to continue at25°C for 48h with stirring. 2-Aminoethanol-Sepha-rose was washed with water.Assay of ribonuclease activity. Ribonuclease acti-

vity was determined by the method of Kunitz (1946).The ribonucleic acid employed in the assay was firstdialysed against several changes of 0.1M-KC1 for48h, precipitated from solution with ethanol,collected by centrifugation and air-dried. A standardcurveforRNAase activitywas obtained with solutionscontaining 2-8,ug of ox RNAase/ml.

Fractionation ofRNAasefrom Bison bisonpancreasglands. The procedures of McDonald (1956) werefollowed in part. Pancreas glands were obtained fromBison bison that had been recently slaughtered duringa herd reduction programme in Elk Island NationalPark, Alberta, Canada. The glands were removed inan abattoir and quickly frozen on solid CO2. Theywere kept at -20°C until required. Eleven pancreasglands were thawed in 0.125M-H2SO4 in the cold-room overnight. Fat and connective tissue were re-moved and the glands were minced in an electric meat

grinder to yield a volume of 2 litres. Cold 0.125M-H2SO4 (4 litres) was added and the suspension wasstirred overnight in the cold-room. The suspensionwas strained through cheesecloth and the residue wasresuspended in 1 litre of 0.125M-H2SO4 and furtherextracted for 2h. The extracts were strained throughcheesecloth again, then pooled to yield a volume of5 litres with an absorbancy at 280nm of 0.30 for a1:400 dilution with water.By using a nomogram for obtaining amounts of

(NH4)2SO4 salt to be added (Dixon, 1953), fractiona-tion of the H2SO4 extract was made between 0-20%,20-40%, 40-65% and 65-85% saturation. The resi-due remaining from the latter fractionation (the onlymaterial used in these studies) was dissolved in 100mlof water, subjected to Millipore filtration to removeparticulate matter and desalted on a column(4cm x 77cm) of Sephadex G-25 (medium grade) at4°Cwithwater as eluant. Proteinfractions werepooledand freeze-dried. The freeze-dried material was dis-solved in 0.025M-piperazine adjusted to pH 5.3 with0.5M-HCI and was applied to a NH2pPUP-Sepha-rose affinity column as outlined in Fig. 3. Elution ofmaterial absorbing at 280nm was continued withpiperazine-HCl buffer until the absorbancy readingapproached 0.03 when the piperazine-HCl wasreplaced with 0.25M-sodium phosphate buffer, pH3,to desorb the affinity-bound bison ribonuclease A.

Preparation ofbison carboxymethyl-RNAase. BisonRNAase purified by Bio-Rex-70 chromatographywas desalted on Sephadex G-25 and freeze-dried.Inhibition by iodoacetic acid was performed as out-lined by Crestfield et al. (1963a). To a solution ofbison RNAase (40mg, 2.8,mol in 3ml of water) atpH 5.5 was added 1 ml of iodoacetic acid (5.3mg,28.6,umol) previously adjusted to pH 5.5. The inhibi-tion was conducted in a pH-stat at 25°C under N2and in the dark with the pH being maintained atpH5.5 by the addition of 0.1M-NaOH. Assays per-formed during the inhibition indicated that 37% ofthe activity remained after 7h, 21 % remained after28h and zero activity after 70h. The reaction mixturewas desalted on a column (2cm x 82cm) of SephadexG-25 (fine grade) equilibrated and eluted with 50mM-acetic acid. The column was wrapped with aluminiumfoil to exclude light. Monitoring the effluent by280nm absorbancy and conductivity measurementsindicated one E280 peak eluting just after the voidvolume with very low conductivity and a secondE280 peak eluting at twice the void volume with highconductivity. The fractions corresponding to eachpeak were freeze-dried. The early eluting peak wassubsequently identified as His(Cm)RNAase (25mg),whereas the retarded peak contained iodoacetic acidand salts.

Preparation of bison RNAase labelled covalentlywith pyridoxal 5-phosphate. Bison RNAase (20mg)was treated with 0.69mg of pyridoxal 5-phosphate in

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RIBONUCLEASE A FROM BISON BISON

0 20 40 60 0

Fraction number20 40 60

Fig. 1. Elution profiles ofchymotrypsin a andox ribonuclease on unsubstituted Sepharose-4B

(a) Chymotrypsin a (4mg) was chromatographed on a column (0.9cm x 5cm) of unsubstituted Sepharose-4Bequilibrated and eluted with 0.02M-sodium acetate buffer, pH 5.2, at 250C. The flow rate was 30ml/h with 1 mlfractions being collected. The arrow (4) indicates where elution with 0.2M-acetic acid commenced. (b) Chymo-trypsin a (4mg) was chromatographed as in (a) but the column was equilibrated with 0.025M-piperazine-HClbuffer, pH 5.3, at 25°C. The arrow (4) indicates where elutionwith 0.2M-acetic acid commenced. (c) Ox ribonuclease(4.2mg) was chromatographed on a column (0.9cmx5cm) of unsubstituted Sepharose-4B equilibrated andeluted with 0.02M-sodium acetate buffer, pH5.2, at 25°C. The flow rate was 30ml/h with ml fractions beingcollected. The arrow (4) indicates where elution with 0.2M-acetic acid containing 0.2M-KCI commenced. (d) OxribonucleaseA (4mg) was chromatographed on a column (0.9cm x 5cm) of Sepharose-4B equilibrated and elutedwith 0.025 M-piperazine-HCl buffer, pH5.3, at 25°C. The flow rate was 26ml/h with 1 ml fractions being collected.The arrow (4) indicates where elution with 0.2M-acetic acid commenced.

4ml of 0.05M-Tris adjusted to pH7.0 with 0.5M-acetic acid as described by Means & Feeney (1971).NaBH4 was added until the yellow colour of the solu-tion disappeared. The coupling was repeated with0.54mg of pyridoxal 5-phosphate followed by re-duction with NaBH4. The reaction products wereseparated on a column (2cm x 82cm) of SephadexG-25 (fine grade) equilibrated and eluted with 50mM-acetic acid at 25°C. The flow rate was 30ml/h with afraction size of 4m1. The elution pattern was moni-

Vol. 135

tored at 280nm to detect protein and at 324nm todetect the pyridoxal 5-phosphate chromophore. Apeak with fractions possessing similar absorbancyreadings at 280nm and 324nm eluted at about 1.2void volumes and was collected and freeze-dried(recovery 16mg). A spectrum of this material showedthe absorbancy maxima at 270nm and 328nm ex-pected for a pyridoxal 5-phosphate-RNAase. Basedon an extinction coefficient at 324nm of 7700 litre-mol1 cm-' for reduced pyridoxal 5-phosphate

000

429


attached covalently to RNAase, the ratio ofpyridoxal5-phosphate to bison RNAase was calculated to be1.28. A second peak with very high 324nm and low280nm absorbancy was eluted with about 2 void vol-umes of acetic acid. This material contained salts andreduced pyridoxal 5-phosphate. Fractions of a thirdpeak possessing high 290nm and low 324nm absorb-ancy readings were pooled and freeze-dried butyielded an oily residue. The nature of this material isunknown.

High-voltage electrophoresis. The vertical strip high-voltage electrophoresis apparatus was similar to thatdescribed by Michl (1951) as modified by Ryle et al.(1955). The buffer systems and coolants used were asdescribed by Smillie & Hartley (1966). The two-dimensional high-voltage electrophoresis isolation of

the pyridoxal 5-phosphate-labelled peptide wascarried out in pH 1.6 buffer (8.6% formic acid) andpH2.4 buffer (4% formic acid, 0.58% pyridine)(K. J. Stevenson, unpublished work). Peptides weredetected with the cadmium-ninhydrin dip reagent ofHeilmann et al. (1957) by developing guide strips ofchromatograms, and amino acids were detected witha dip reagent consisting of0.7ml ofcollidine in 100mlof0.5% ninhydrin in acetone. The presence oftrypto-phan-containing peptides was investigated with Ehr-lich reagent (p-dimethylaminobenzaldehyde) as des-cribed by Bailey (1968). The suitability of the Ehrlichreagent was confirmed by obtaining positive resultswith peptides known to contain tryptophan. Visualdye markers were used throughout high-voltageelectrophoresis work as described by Stevenson

0.

0.

0.

0Go

0.

0.

0.

,6 - (a) (b)

.2 1 2 1 2 3

C ~~~I II I

(c) (d)

.6-

.4-

2 ~~ ~~~~2

0 40 80 120 0 40 80 120Fraction number

Fig. 2. Elutionprofiles ofox ribonuclease andchymotrypsin oc on NH2pPUP-Sepharose(a) Ox ribonuclease A (4mg) was chromatographed on a column (0.9cm x 20.5 cm) of NH2pPUP-Sepharose in0.02M-sodium acetate buffer, pH 5.2, at 25°C. The flow rate was 26ml/h with fraction size of t ml. The arrow (U1)indicates elution with 0.2M-acetic acid, the arrow (U2) elution with 0.2M-acetic acid containing 0.2M-KCI and thearrow (U3) elution with 0.2M-KCI adjusted to pH2.0 with HCI. (b) Chymotrypsin a (4mg) was chromatographedon NH2pPUP-Sepharose as outlined in (a). (c) Ox ribonuclease A (4mg) was chromatographed on acolumn (0.9cm x4.5cm) of NH2pPUP-Sepharose in 0.025M-piperazine-HCl buffer, pH 5.3, at 25°C. The flowrate was 26ml/h with fraction size of 1 ml. The arrows (I1), (42) and (43) indicate elutions as outlined in (a).(d) Chymotrypsin o (4mg) was chromatographed on a column (0.9cmx5cm) of NH2pPUP-Sepharose in0.025M-piperazine-HCI buffer, pH 5.3, at 25°C. The flow rate was 26ml/h with fraction size of 1 ml. The arrowU,) indicates elution with 0.2M-acetic acid.

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(1971). Homoserine lactone was prepared by heatinghomoserine in 6M-HCI for 2h at 110°C in a sealed,evacuated tube. Carboxymethylhistidine derivativeswere synthesized as outlined by Crestfield et al.(1963a) and were separated on a column (1.2cmx30cm) of Dowex 50 (X8) in the pyridinium form. Agradient of 200ml of 0.1 M-pyridine-acetic acid,pH3.2, and 100ml of 2.1 M-pyridine-acetic acid,pH6.0, was employed. Final purification was ob-tained on high-voltage electrophoresis at pH6.5(cf. Fig. 4).

Diagonal peptide 'mapping' of bison and ox ribo-nuclease. Diagonal peptide 'mapping' was carriedout as described by Brown & Hartley (1966). BisonRNAase (20mg) was digested with 2mg of pepsin in5mlof 5% (v/v)formicacidat 28°C for 16h. The digestwas spotted on Whatman 3MM paper as a 25cmband at a distance of 12cm from the anode and wassubjected to high-voltage electrophoresis at pH3.5and 3kV for 1 h. A 3cm side-strip from the initialchromatograms was placed in a desiccator andoxidized by performic acid vapour. The diagonalpeptide 'map' of peptides containing cysteic acid wasformed by subjecting the oxidized side-strip to high-voltage electrophoresis at pH3.5 and 3kV for 1 h(cf. Fig. 5). This 'map' served as the basis for thesubsequent isolation of peptides containing cysteicacid from the initial high-voltage run at pH3.5.Cysteic acid-containing peptides were purified byhigh-voltage electrophoresis at pH3.5 based on thediagonal peptide 'map' obtained at that pH (cf. Fig.5). All ofthe peptides were cleanly separated from oneanother and only peptide B4 was subjected to furtherpurification by high-voltage electrophoresis at pH2.0(3kV for 3 h).

Isolation ofpyridoxal 5-phosphate-labelled peptidefrom bison RNAase-pyridoxal 5-phosphate. After per-formic acid oxidation (Hirs, 1956) of bison RNAase-pyridoxal 5-phosphate, the freeze-dried material(14.5mg) was digested with porcine trypsin in a pH-stat at pH8 and 25°C for 16h. The digest was appliedas a 19cm band at a distance of 10cm from the anodeon Whatman 3MM paper and subjected to high-voltage electrophoresis at pH 1.6 (2kV, 1 h). A majorfluorescent ninhydrin-positive band was observedwith a mobility of ms," = 0.36. Other minorfluorescent bands were also noted. The major fluor-escent band was cut out, sewn on to a second sheet of3MM paper at a distance of 12cm from the anodeand subjected to high-voltage electrophoresis atpH2.4 (2kV, 1 h). A single fluorescent peptide withan orange, changing to pink, cadmium-ninhydrincolour was obtained with a mobility of ,n2e' = 0.55.The peptide was eluted with 1.3ml of 10mM-aceticacid and the absorbancies of this solution were 0.40and 0.65 at 280nm and 324nm respectively.Amino acid analyses. The amino acid analyses of

RNAase A from Bison bison were performed by

Vol. 135

hydrolysing 3.0mg samples of RNAase in 1 ml of6M-HCl at 110°C for 18, 24,48 and 72h. To each test-tube was added 59.2,utg of norleucine to act as aninternal standard (Walsh & Brown, 1962). The acidhydrolysates were rapidly evaporated to dryness on aEvapomix and were analysed on a Beckman 120Camino acid analyser by the methods of Spackmanet al. (1958).The quantitation ofcystine as cysteic acid after per-

formic acid oxidation by the method of Hirs (1956)was determined on a Phoenix M-7800 amino acidanalyser.The quantitation of N'-carboxymethylhistidine

and NM-carboxymethylhistidine was carried out onperformic acid-oxidized bison His(Cm)RNAase bythe method of Crestfield et al. (1963a) employing thePhoenix analyser. Performic oxidation was necessary

1.6

1.4-

1.2-

1.0-

o 0.8

0.6

0.4

0.2

l0 20Fraction number

Fig. 3. Selective isolation ofbison ribonucleasefrom acrudepancreatic extract by affinity chromatography on

NH2pPUP-SepharoseThe sample was 200mg of a crude desalted prepara-tion derived from bison pancreas glands (see theExperimental section). Affinity chromatography wasconducted on a column (0.9cmx 13.3cm) ofNH2pPUP-Sepharose equilibrated and eluted with0.025M-piperazine-HCl buffer, pH5.3, at 25°C.The flow rate was 30ml/h with 5ml fractions beingcollected. The arrow (;) indicates where elution com-menced with 0.25 M-sodium phosphate buffer, pH 3.

431


to transform cystine into cysteic acid, thus eliminatingthe overlap on the elution profile between cystine andN?-carboxymethylhistidine.

Results and DiscussionIsolation ofbison ribonucleaseDuring the application of affinity chromato-

graphy on NH2pPUP-Sepharose for the selective iso-lation of bison RNAase by the method of Wilchek &Gorecki (1969), difficulties were encountered in tryingto obtain successful application of this elegant tech-nique for enzyme purification. The areas of concernwere ultimately traced to (1) the existence of nega-tively charged species (likely carboxyl groups, in-herent in the native unsubstituted Sepharose-4B) and(2) the inability of 0.2M-acetic acid to remove theadsorbed RNAase from the NH2pPUP-Sepharoseaffinity column.To obtain evidence for the presence of low concen-

trations of carboxyl groups in native Sepharose-4B,ox RNAase and bovine chymotrypsin a were chro-

matographed separately in the presence of 0.02M-acetic acid adjusted to pH 5.2 with 1M-NaOH and0.025M-piperazine-HCl buffer, pH5.3, as shown inFig. 1. Equilibration and elution of unsubstitutedSepharose with the acetate buffer caused both pro-teins to be weakly adsorbed to the column as indi-cated by the lengthy tailing of the peaks (Figs. la andlc). After the stepwise addition of 0.2M-acetic acid,to desorb any protein material not eluted with acetatebuffer, portions of chymotrypsin a and ox RNAasewere further desorbed from the Sepharose as shownin Figs. 1(a) and 1(c), respectively. The results clearlysuggest the existence of non-specific interactions be-tween the Sepharose matrix and protein molecules.The nature of these interactions is predominantlyionic and probably involves carboxyl groups onSepharose (Porath etal., 1971). The existence of thesenegatively charged species was supported by theobservation that pepsin, an acidic protein with a plof 2, passed unretarded through Sepharose underconditions similar to Fig. 1(a). Moreover, Sepharose,in which the majority of the carboxyl groups had been

Table 1. Amino acid composition ofbison ribonuclease A

The value for alanine is arbitrarily set at 12.0 residues, for an average of three analyses per hydrolysis time. Thevalues for ox were reported by Smyth et al. (1963).

Amino acid composition

Amino acidLysineHistidineArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystine(Cysteic acid)ValineMethionine(Methionine

sulphone)IsoleucineLeucineTyrosinePhenylalanineTryptophan

Hydrolysistime ...

Average18h 24h 48h 72h value10.4 11.1 10.7 10.5 10.73.9 4.2 4.3 4.1 4.14.1 4.1 4.3 4.1 4.1

15.3 15.4 15.4 15.3 15.49.3 9.5 8.9 8.9 9.8*

13.1 12.1 11.5 8.9 14.6*12.2 11.9 12.3 12.2 12.24.4 3.8 4.1 4.1 4.13.2 3.2 3.2 3.2 3.212.0 12.0 12.0 12.0 12.09.4 9.7 9.3 9.0 9.4

(7.0)§8.7 9.1 9.3 9.2 9.24.2 4.1 4.2 4.1 4.2- - - - (4.0)§

Integralvalue11441510151243129(8)94(4)

Ox10441510151243128

(8)94(4)

2.2 2.7 3.0 3.0 3.Ot 3 32.2 2.2 2.2 2.2 2.2 2 26.0 6.1 6.0 5.9 6.0 6 63.2 3.2 3.3 3.4 3.3 3 3- - - - 0 01 0

* Extrapolated to zero time.t Value for the 72h hydrolysis time was taken.$ Based on Ehrlich's reagent test for tryptophan on two-dimensional peptide 'maps'.§ Average of three analyses for oxidized bison RNAase. Cysteic acid was corrected for 94% recovery (Moore, 1963).

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transformed into uncharged -CO-NH-CH2CH20H moieties by covalent attachment of 2-amino-ethanol (see under 'Methods'), allowed both chymo-trypsin ac and ox RNAase to pass through essentiallyunretarded. This was in contrast with the elutionprofiles of these enzymes on native (unsubstituted)Sepharose (cf. Figs. la and Ic).Piperazine-HCl buffer was substituted for sodium

acetate buffer in an attempt to eliminate non-specificinteractions between the suspected negatively chargedgroups on the Sepharose matrix and protein mole-cules under study. The positively charged heterocyclicring of piperazine was considered to be a more effec-tive competitor than the sodium ion for these groups.This view was based on observations that pyridiniumions effectively decreased tailing of chromatographicpeaks eluting from sulphonate polystyrene resins(Dixon, 1966). Sodium ions were much less effectiveat decreasing tailing than were pyridinium ions atsimilar concentrations.

The rationale for employing a piperazine-HClbuffer was supported by the data in Figs. l(b) andl(d). Both chymotrypsin and RNAase A passedunretarded through Sepharose when this buffer wasemployed, but were weakly adsorbed when sodiumacetate bufferwas used. Thesum ofthe concentrationsof monovalent and divalent piperazinium ions(10mM and 15mM respectively) was about twice theconcentration of the sodium ion (14mM) present inthe acetate buffer. The greater effectiveness of piper-azine-HCl buffer in competing for the negativecharges on Sepharose was due, not only to the highertotal concentration of cations present, but also to theprobable non-ionic interactions of the monovalentand divalent piperazinium ions with the Sepharosematrix.

Control affinity-chromatography runs were per-formed on NH2pPUP-Sepharose with ox RNAaseA as the specific enzyme and bovine chymotrypsin asa non-specific enzyme. RNAase was adsorbed to

- Hse>

o Lys C_ Arg,

C=: His c_>

His(Cm)RNAase

S S A B C M S S

IlIlIllIllR 111111111°

(:2:::)cO Hse

XCFF

| sOG ~~~~CmCyFz OC=>Glu C* C

° C C Asp b- > Cys(03H) +

Fig. 4. Chromatogram ofa qualitative amino acidanalysis ofan acidhydrolysate ofbison His(Cm)RNAaseHigh-voltage electrophoresis was conducted at pH6.5 and 3kV, until the dye marker Orange G (OG) migrated19cm towards the anode. Amino acid and dye markers are indicated by S; XCFF refers to Xylene Cyano FF. Adenotes Nl-'?dicarboxymethylhistidine, B denotes N?-carboxymethylhistidine, C denotes N'-carboxymetbyl-histidine and M denotes a mixture of the three carboxymethylhistidine derivatives.Vol. 135

433


Lys

c MGc_) Arg o

B2~6

)B"' 0A Dnp-

cDZ3A2 TauLHOrigin ° BS -)A3

N C )~~~~~~~A5f Asp

t XCFF +

cvQ

Gly (

Ser e_-Lys C:rine r-:>

Fig. 5. Diagonalpeptide 'map' ofapepsin digest ofbison ribonuclease

High-voltage electrophoresis was conducted at pH3.5 and 3kV for 1 h in both dimensions. N denotes a mixture ofamino acids and visual dye markers. MG and CV denote Methyl Green and Crystal Violet respectively. XCFFrefers to Xylene Cyano FF and A1-A5, B1-B5 and Cl are peptides (see Table 3).

N

NH2pPUP-Sepharose when either acetate buffer orpiperazine buffer was used. However, desorption ofRNAase from the affinity column was very difficult,particularly when the enzyme had been applied withacetate buffer. As shown in Fig. 2(a), the stepwiseaddition of 0.2M-acetic acid failed to desorb the en-zyme initially applied in acetate buffer and the addi-tion of 0.2M-acetic acid containing 0.2M-KCl wasrequired to remove, albeit slowly, the RNAase fromthe column. Additional RNAase was removed whenthe more rigorous eluent, 0.2M-KCI adjusted topH 2.0 with HCI, was employed. The use ofpiperazinebuffer to replace acetate buffer did improve the elutionproffle: during desorption, about one-half of theRNAase was eluted with 0.2M-acetic acid and theremaining material bound on the column was de-sorbed with 0.2M-acetic acid containing 0.2M-KCI(Fig. 2c). However, satisfactory desorption was notobtained with this system.

Substantial non-specific adsorption of chymotryp-sin a occurred when acetate buffer was used duringchromatography on NH2pPUP-Sepharose. In this

system, marked retardation was observed in the pre-sence ofacetate buffer and the complete desorption ofchymotrypsin a required potent elution systems (Fig.2b). By employing piperazine buffer to apply theenzyme, all of the chymotrypsin a passed through theNH2pPUP-Sepharose column unretarded (Fig. 2d)in marked contrast with the experiments employingacetate buffer. The work with chymotrypsin a servedto indicate the importance of masking chargedcarboxyl groups on the Sepharose matrix and mask-ing the negatively charged phosphate moiety intro-duced with NH2pPUP to improve the biologicalspecificity of the affinity column employed.The disparity between the present data and the

results of Wilchek & Gorecki (1969) is undoubtedlydue to the increase in the column size (up to 13-fold)used in these studies, reflecting the difficulties thatcan arise through scaling-up of procedures.

After experiments with various reagents, desorp-tion of affinity-bound RNAase from NH2pPUP-Sepharose was ultimately obtained quickly and com-pletely through the replacement of the acetic acid and

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434


Table 2. Amino acid composition ofpeptides isolated by diagonal peptide 'mapping' of bison ribonuclease A

The numbering of the peptides is explained in Fig. 5. Peptide B4 was further purified on high-voltage electropho-resis at pH2.0 (3kV for 3h).

PeptideAl (minor)

A2 (major)

A4 (major)

A5 (minor)BI (intermediate)

B3 (intermediate)

B4 (major)

B5 (major)

Cl (major)

Cadmium-ninhydrincolour Composition

Yellow -* red Cys(03H) (1.0), Asp (1.0)*, Thr (1.2), Ser (3.4), Glu (0.9), Pro (+)t,Gly (1.2), Ala (0.9), Tyr (0.2), Lys (0.6), Arg (0.5)

Red Cys(03H) (1.6), Asp (1.9), Thr (2.3), Ser (2.4), Glu (1.0)*, Pro (+)t,Gly (1.0), Ala (0.8), Ile (1.0), Tyr (0.9), Lys (0.9), Arg (1.0)

Red Cys(03H) (0.8), Asp (1.9), Ser (2.6), Glu (1.0)*, Ala (0.8),Met(02) (1.2), Tyr (0.9)

Red Cys(03H) (0.8), Asp (1.1), Ser (0.3), Glu (0.9), Met(02) (1.0)*, Tyr (0.8)Red Cys(03H) (0.5), Asp (1.7), Thr (0.7), Ser (1.0), Glu (2.0)*, Gly (0.8),

Ala (0.9), Val (1.0), Tyr (1.1), Lys (0.3)Red Cys(03H) (1.8), Asp (3.0)*, Thr (0.9), Ser (2.0), Glu (3.1), Gly (1.4),

Ala (2.1), Val (2.4), Tyr (1.2), Lys (0.9)Yellow orange Cys(03H) (0.8), Asp (1.0)*, Glu (1.0), Pro (+)t, Gly (1.1), Val (1.5),

Tyr (1.0), Phe (1.2), His (1.0)Red Cys(03H) (2.5), Asp (3.4), Thr (1.3), Ser (2.3), Glu (3.4), Gly (1.0),

Ala (2.0)*, Val (2.2), Tyr (1.6), Lys (0.3)Yellow orange Thr (0.9), Ser (0.8), Met(02) (1.0)*

* Arbitrarily taken as 1.0, 2.0 or 3.0 residues as indicated with recoveries of other amino acids relative to this amino acid.t Detected on the amino acid analyser but not quantified owing to instrument malfunction.

acetic acid-KCl solutions with 0.25M-phosphoricacid adjusted to pH 3.0 with 1 M-NaOH. Since phos-phate ion at pH5.2 is an effective inhibitor of RNA-ase (Wyckoff et al., 1967), it was felt that affinity-bound RNAase could be desorbed from NH2pPUP-Sepharose with 0.25M-sodium phosphate buffer atpH 3.0 because of the combined effects of low pH,high ionic strength and competitive binding of theH2PO3- ion.The selective isolation of Bison bison RNAase

from crude pancreatic extracts is presented in Fig. 3.NH2pPUP-Sepharose specifically adsorbed RNAasefrom a heterogeneous mixture of proteins chromato-graphed in 0.025M-piperazine-HCl buffer, pH5.3.Continual elution with piperazine buffer removed allextraneous proteins but did not lead to removal ofbound RNAase. Desorption of the RNAase wasaccomplished by the stepwise addition of 0.25M-sodium phosphate buffer, pH3.0. The purity of thispreparation of bison RNAase was attested to bysubsequent ion-exchange chromatography on Bio-Rex-70 (Hirs et al., 1953). Only one major peak pos-sessing RNAase activity was evident and this material,after desalting on Sephadex, was employed in allsubsequent characterizations of the enzyme.

Characterization ofbison ribonuclease A

The amino acid composition of ribonuclease fromBison bison is shown in Table 1 along with the amino

Vol. 135

acid composition of ox RNAase. The data are basedon assuming a value of 12.0 residues of alanine forbison RNAase and indicate the apparent similaritybetween the ox and bison enzymes, which are iso-lated from animals within the Bovidae family. Theonly amino acid that appears to vary is lysine; thereis one more residue present in the bison RNAasethan in the ox RNAase. The recoveries of 9.0 residuesof half-cystine are believed not to be reliable. How-ever, quantitation of half-cystine as cysteic acid alsoproved to be unsatisfactory as recoveries were con-sistently low even with ox RNAase taken as a stan-dard. Certainly, the recoveries of 7.0 residues ofcysteic acid in bison RNAase is not a true value, as itimplies the presence of a free thiol group. Aminoacid analyses ofbisonHis(Cm)RNAase did not revealthe presence of any S-carboxymethylcysteine deriva-tives which, if a cysteine residue had been present andaccessible in the native enzyme, would have beenformed preferentially when bison RNAase was ex-posed to iodoacetic acid. The strongest experimentalevidence for assigning a value of eight to the half-cystine content of bison RNAase comes from thediagonal peptide 'maps' of ox and bison RNAaseprepared in three different high-voltage electropho-resis buffer systems (pH2.0, 3.5 and 6.5). These 'maps'were identical in appearance and make it unlikely thatthere was an extra disulphide bridge in the bisonenzyme. Further support for a half-cystine contentof eight residues was obtained by the isolation and

435

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1973


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Vol. 135

437


characterization of peptides containing cysteic acid(cf. Tables 2 and 3). The absence of tryptophan frombison RNAase is based on the specificity of dimethyl-aminobenzaldehyde (Ehrlich's reagent) for the indolering of tryptophan when employed as a detectionreagent on two-dimensional peptide 'maps'. TheEhrlich test for tryptophan was always negative.The extent ofmodification ofthe active site ofbison

RNAaseby iodoacetic acid was determined by analys-ing the acid hydrolysate of three performate-oxidizedand two unoxidized His(Cm)RNAase preparationson an amino acid analyser as described by Crestfieldet al. (1963a). Performic acid oxidation of His(Cm)-RNAase transformed half-cystine into cysteic acidthereby allowing N-carboxymethylhistidine to beeluted in a position normally occupied by half-cystine. The recoveries of N"-carboxymethylhistidineand N-carboxymethylhistidine were 0.62 and 0.36residues respectively, by employing the recovery ofalanine as 12.0 residues and utilizing an averageamino acid integration constant for N'- and N?-carboxymethylhistidine. These derivatives were alsoshown to be present in bison His(Cm)RNAase bysubjecting the acid hydrolysate to high-voltageelectrophoresis at pH6.5 in the presence of syntheticcarboxymethylhistidine standards (Fig. 4). The dataindicate that the modifications of the histidine resi-dues have been mutually exclusive since the net modi-fication ofhistidine is 0.98 residues. The data supportthe finding that the bison His(Cm)RNAase wasenzymically inactive when assayed against RNA. Thedistribution of the modifications in N'- and Nr-

carboxymethylhistidine in bison RNAase is 2:1,whereas Crestfield et al. (1963a,b) have reported thedistribution in ox RNAase to be 8:1 respectively.Since the inactivation was carried out under identicalexperimental conditions, the quantitation suggestsdifferences between these two very closely relatedenzymes. However, more studies are required beforedifferences in active-site reactivity can be clearlyascertained.Diagonal peptide 'maps' of the disulphide bridges

of bison RNAase and ox RNAase were prepared asdescribed by Brown & Hartley (1966) byhigh-voltageelectrophoresis at pH2.0, 3.5 and 6.5. The diagonalpeptide 'maps' of both enzymes prepared at the threepH values were identical in appearance and suggestedclosely related primary structures adjacent to disul-phide bridges. The best separation of the cysteic acid-containing peptides on the diagonal peptide 'maps'was obtained by high-voltage electrophoresis atpH3.5 (Fig. 5).The amino acid composition, cadmium-ninhydrin

colour and the relative recoveries of cysteic acid-containing peptides from bisonRNAase are presentedin Table 2. Peptides referred to as 'major peptides'were recovered in about 80nmol quantities, whereasminor peptides were recovered in 10-2Snmolamounts. Based on the above data, the best position-ing of the peptides into the known amino acidsequence of ox RNAase is shown in Table 3. Thepeptides derived from bison RNAase had extensivehomologies with certain amino acid sequencesadjacent to disulphide bridges of ox RNAase. The

Table 4. Peptides isolatedfrom two-dimensionalpH1.6 andpH2.4 high-voltage electrophoresis of a trypsin digestofbison RNAase-pyridoxal 5-phosphate

Peptide composition

Lys(0.7),Ser(O.9),Arg(l.0)*

Lys(l.9),Thr(1.2),Glu(0.9),Ala(3.0)* Ox:

Peptide sequence alignment31 32 33

Ox: Lys-Ser-ArgBison: (Lys,Ser,Arg)

1 7Lys-Glu-Thr-Ala-Ala-Ala-Lys

Bison: (Lys,Glx,Thr,Ala,Ala,Ala,Lys)

Glu(l.O)*,Phe(l.O),Arg(O.9) Ox:8 10

Phe-Glu-ArgBison: (Phe,Glx,Arg)

Asp(l.O)*,Thr(l.O),Leu(l.O),Lys(l.0) Ox:35 38

Leu-Thr-Lys-AspBison: (Leu,Thr,Lys,Asx)

* Arbitrarily taken as 1.0 or 3,0 residues as indicated with recoveries ofother amino acids relative to this amino acid.

1973

438


Table 5. Characterization ofthefluorescentphosphorylpyridoxalpeptidefrombison RNAase-pyridoxal 5-phosphate

The recovery of the basic compound 'X' was calculated by using an average amino acid integration constant. Thiscompound is the acid-degradation product of E-pyridoxaminolysine, which was eluted from a basic column(0.9cm x 14cm) of the Phoenix analyser with 0.20M-sodium citrate buffer, pH5.28, 55°C, at the 50min position,whereas lysine, histidine and arginine were eluted at 49.5, 57.5 and 108min respectively. The notation Lys indicates

PLPthe site of covalent attachment of pyridoxal 5-phosphate to the isolated peptide.

Amino acid compositionCys(03H)(0.6),Asp(l.O)*,Thr(O.9),Pro(O.3),Val(O.6),Phe(O.6),'X'(0.5)

Sequential alignment03H140 41 45

Ox: Cys-Lys-Pro-Val-Asn-Thr-Phe03H

Bison: (&ys,Lys,Pro,Val,Asx,Thr,Phe)

PLP

* Arbitrarily taken as one residue.

recovery of lysine from the amino acid analyses ofpeptides B1, B3 and B5 was consistently lower thanexpected if total homology existed between thesesegments of both ribonucleases. The possibility existsthat the lysine residue known to be present at position61 (or perhaps position 66) in ox RNAase could bedeleted in bison RNAase.The amino acid composition of peptide A4 from

bison RNAase, isolated in high yield, can be onlyaccommodated with the known amino acid sequenceofresidues 20-29 ofox RNAase. Thus, on the basis ofpeptide characterizations presented, the amino acidsequence adjacent to half-cystine in position 26 inbison RNAase could tentatively be assigned: Ala20-Ser-Ser-Ser-Asn-Tyr-Cys(03H)-Asn-Gln-Met(02)29.Barnard et al. (1972) have reported the amino acidsequence of this region of the bison RNAase to beAla20- ?-Thr-Ala-Asn-Tyr-Cys(03H)-Asn-Gln-Met-(02)29 based on sequential degradation ofthe oxidizedenzyme in a Beckman Protein Sequencer. Identifica-tion of each amino acid residue released was deter-mined on its phenylthiohydantoin derivative by g.l.c.(with and without silylation) and also on t.l.c. Theuncertainty existing in the nature ofthe amino acid atposition 21 (?) has been suggested to arise from acarbohydrate attachment through an asparagineresidue. In this study, the amino acid analyses ofpep-Vol. 135

tide A4 did not indicate the presence of threoninenor did it suggest the existence ofan amino sugar. Thelatter could have been formed during acid hydrolysisand would be expected to be partially stable. The dis-parity between the data is probably not due to dif-ferences in subspecies within the genus Bison, sincethe American or Plains bison (Bison bison bison) wasundoubtedly utilized by both groups. RNAase fromthe closely related Wood bison (Bison bisonathabascae) would not have been studied, since thisanimal is considered rare with only a few animalsexisting within the National Parks of Canada.

Support for the published sequence ofthe 30N-ter-minal residues of bison RNAase (Barnard et al.,1972) was obtained by the isolation of peptides pos-sessing the amino acid composition equivalent to thesequences Lys'-Glu-Thr-Ala-Ala-Ala-Lys7 and Phe-8Glu-Arg10 (Table 4).The modification of ox RNAase with pyridoxal 5-

phosphate followed by reduction with NaBH4 wasreported by Means & Feeney (1971) to be associatedwith the covalent attachment to the c-amino group oflysine-7. However, studies by Raetz & Auld (1972)revealed that in addition to lysine-7, lysine-41 wasalso modified during the reaction of equimolar mix-tures of ox RNAase and pyridoxal 5-phosphate.Amino acid analyses of phosphorylpyridoxal pep-

439


Table 6. Bison ribonuclease disulphide peptides

The pairing of the cysteic acid peptides isolated from the pH3.5 diagonal peptide 'map' (Fig. 5) is based on datain Tables 2 and 3 and on the known order for ox RNAase disulphide bridges: 26-84, 40-95, 58-110 and 65-72.The peptide A. was not recovered and its proposed sequence is arbitrarily based on the specificity of pepsin. Pep-tide Cl did not contain cysteic acid but was located below the diagonal as a result of the formation of methioninesulphone, which increased the molecular weight of the peptide by 8% and could have influenced the pK of thea-CO2H group.

Group A36 40 46Thr-Cys-Phe

SS

85 X95 96Arg-Cys-Ala

81 84 95 96Ile-Cys-Cys-Ala

SS

25 \ 26 29Tyr-Cys-Met20 26 29Ala-Cys-Met

Group B

Peptide

A,

Al

A2

A5

A4

S-S63 165 172 75Val-Cys-Cys-Ser

S -S56 58 165 172 75Ala-Cys--Cys-Cys-Ser55 58 65 72 76Gln-Cys-Cys--Cys-Tyr

IS S--SShlo 120Cys-Phe

BI

B3

B5

B4

Group C78 79 80Thr-Met-Ser

02

Thr-Met-Ser

(before oxidation)C1

(after oxidation)

tides indicated that lysine-7 and lysine-41 were modi-fied in a ratio of 2:3. Bison RNAase, covalentlymodified with pyridoxal 5-phosphate under condi-tions employed by Means & Feeney (1971) for oxRNAase, yielded a single highly fluorescent peptideafter two-dimensional high-voltage electrophoresisofa trypsin digest. Amino acid analysis ofthis peptidewas only compatible with the amino acid sequence ofoxRNAase adjacent to lysine in position 41 (Table 5).

Other very weakly fluorescent peptides were observedafter high-voltage electrophoresis but attempts to iso-late these were unsuccessful because of the limitedamounts of the peptides.On the basis of the amino acid composition of

peptides isolated from bison RNAase, almost com-plete homology with the corresponding segments ofoxRNAasewas observed. Ofthe 90residues for whichthe information exists, 89 appear to be homologous

1973

440

RIBONUCLEASE A FROM BISON BISON 441

when side-chain amide groups are assigned on thebasis ofhomology with the ox sequence. The peptidesstudied from bison RNAase essentially representedamino acid residues adjacent to the disulphidebridges, which tend to be invariant segments of pro-tein structure (Table 6). In addition, the bison andthe ox are closely related species within the familyBovidae. The high degree of homology observedbetween the RNAase enzymes from these species istherefore to be expected.

The authors acknowledge the generous assistance ofDr. L. B. Smillie and Mr. Mike Nattris, Department ofBiochemistry, University of Alberta, Edmonton, Alberta,Canada, for amino acid analyses. The co-operation andassistance of Dr. Ann C. Currier of the Canadian Wild-life Service and the National and Historic Parks Branchof Canada is gratefully acknowledged. This research wassupported by the National Research Council of Canadathrough Grant A 5859.

References

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Aqvist, S. E. G. & Anfinsen, C. B. (1959) J. Biol. Chem.234, 1112-1117

Bailey, J. L. (1968) Techniques in Protein Chemistry, 2ndedn., p. 21, Elsevier Publishing Co., London

Barnard, E. A. (1969) Annu. Rev. Biochem. 38, 677-732Barnard, E. A., Cohen, M. S., Gold, M. H. & Kim, J.

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Chem. 238, 227-234Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal.

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Vol. 135

the isolation and partial characterization of ribonuclease a from

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