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A protein subunit of bromegrass mosaic virus
J. Mol. Biol. (1964) 8, 314--323
A Protein Subunit of Bromegrass Mosaic Virus D. STUBBS AND PAUL KAESBERG Department of Biochemistry, University of Wisconsin Madison 6, Wisconsin, U.S.A. JOHN
(Received 11 October 1963) The protein and RNA of bromegrass mosaic virus were separated from each other by dialysis of the virus against 1 M-calcium chloride. In this medium the virus is degraded, the RNA is quantitatively precipitated and there remains a supernatant solution of viral protein. The protein obtained in this way was soluble in various buffers in the pH range 5·5 to 6·5 and exhibited a single symmetrical sedimenting boundary in the ultracentrifuge. Its molecular weight was 40,000 ± 2,000 as determined by the Ehrenberg method of approach to sedimentation equilibrium. Amino acid analyses indicated that there were about 52 lysyl plus arginyl residues per molecular weight of 40,000. Tryptic hydrolysis of ureadenatured protein yielded 23 soluble peptides and an insoluble residue amounting to about 20% by weight of the intact protein. From the number of soluble tryptic peptides and an assumed number of tryptic peptides in the insoluble residue it was concluded that the molecular weight of the protein subunit of bromegrass mosaic virus was slightly greater than 20,000 and that the protein moiety obtained by treatment with 1 M.CaCla was a relatively stable dimer of the subunit. Partial separation of the soluble peptides resulting from tryptic hydrolysis was achieved on a preparative scale by column chromatography.
1. Introduction BMVt is a small, nearly spherical, RNA-containing plant virus. Physical and chemical studies (Bockstahler & Kaesberg, 1962; Anderegg, Wright & Kaesberg, 1963) have shown that the virus particle contains 79% protein and 21% RNA. It weighs 4·6 x 10 6 atomic mass units. Its external diameter is about 260 A. It has a central cavity 80 A in diameter which is surrounded by a shell-shaped region containing the viral RNA, and this in turn is surrounded by a shell of structural protein, presumably in a symmetrical array of subunits. Yamazaki & Kaesberg (1963a) found that BMV is degraded in 1 M-calcium chloride to yield a soluble fraction which is exclusively protein and a precipitate which consists of the viral RNA. This simple procedure yields viral protein in a form suitable for further investigation. Broad bean mottle virus, which is superficially similar to BMV, is similarly degraded (Yamazaki & Kaesberg, 1963b), although in this case the precipitate contains appreciable protein. It is the purpose of this paper to present physical and chemical characteristics of the protein isolated by calcium chloride treatment of BMV.
t
Abbreviation used: BMV = bromegrass mosaic virus. 314
BROMEGRASS :\IOSAIC VIRUS PROTEIN
315
2. Materials and Methods (i) Preparation of BMF protein
BMV was isolated and purified as described by Bockstahler & Kaesberg (1962). The purified virus was treated with 1 M-CaCI2 in the following way. A suspension containing 1% BMV in water was dialysed for 24 hr at 5°C against 1 M-CaCI 2 plus 0·05 :\I-sodium cacodylate, pH 6·0. Approximately I 1. CaCI 2 solution was used for dialysis of 15 m!. of the BMV suspension contained in Visking 18/32 in. dialysis tubing. During dialysis, a flocculent white precipitate formed. The suspension was centrifuged at 10,000 rev./min for 30 min in a Spinco model L no. 40 rotor. The supernatant liquid, which contains the viral protein, was decanted from the pelleted precipitate, which contains the viral RNA, and was dialysed exhaustively against a suitable buffer or water. If such solutions were warmed above 10°C, considerable insoluble material formed. However, the solutions could be stored cold or frozen without loss of protein solubility or evidence of aggregation. Upon lyophilization, a considerable portion of the dried protein would not go back into solution under mild conditions. (ii) Analytical procedures Ultraviolet absorption spectra of BMV protein were obtained with a Cary Recording Spectrophotometer, model 11, equipped with a matched set of quartz cells of 1 cm path length. The spectra in the 230 to 290 mIL region were corrected for light scattering by extrapolation from the neighboring non-absorbing region. Ultracentrifuge analyses were made in the 12 mm synthetic boundary cell of a Spinco model E analytical ultracentrifuge equipped with a phase contrast Schlieren optical system. Sedimentation coefficicnts wcre determined at 59,780 rev.fmin at 4°C. In all runs an ionic strength of 0·1 or greater was maintained in order to swamp out charge effects. Sedimentation coefficients were corrected to standard conditions and are designated sgo,w' Molecular weights were determined by the Ehrenberg method of approach to equilibrium (Ehrenberg, 1957). In this procedure the ratio of the sedimentation coefficient to diffusion coefficient (8/ D) of a sedimenting substance is obtained from the sedimentation pattern near the meniscus of the solution as sedimentation equilibrium is approached. Such a determination involves two runs in one of which the protein boundary is made synthetically. In the experiments reported here, 0'40 m!. of an 0·8% solution of the protein exhaustively dialysod against an appropriate buffer were spun at 19,160 rcv.jmin in the 12 rom synthetic-boundary cell. Four exposures of the refractive index gradient curve were taken, the first, 90 min after reaching speed and the remaining three at 32 min intervals. Temperature was maintained at 1· 1°C. A second run was then made with the same cell and solution (redispersed by gentle shaking) but 0·30 m!. of equilibrated buffer were layered over the sample when the speed of rotation was 5000 to 7000 rev.lmui. In other details, the second run duplicated the first. Values of S/ D were obtained from these patterns and were inserted into the familiar Svedberg equation. The partial specific volume of the solute was taken as 0·74 m!./g (Bockstahler & Kaesbcrg, 1962). (iii) Amino acid anolsjsee For these analyses BMV protein in 1 M-CaCI 2 was dialysed exhaustively against distilled water. Approximately 4 mg of protein were dried and hydrolysed for various t.imes in 0'5 ml. of glass-distilled constant-boiling HCI at 110°C under nitrogen gas. The amino acid composition of the hydrolysates were determined on a Spinco model 120 automatic amino acid analyser as described by Spackman, Stein & Moore (1958). Cysteic acid was detennined on a protein sample oxidized with performic acid according to the method of Hirs (1956). Tryptophan was estimated from the ultraviolet absorption spectra of the protein in 0·1 N·NaOH according to the method of Goodwin & Morton (1946). (iv) Tryptic digestion Salt-free trypsin, twice crystallized (Worthington Biochemical Sales Corp.) was treated with 0·0625 N-HCI at 37°C for 18 hr to lower preferentially a possible contaminating chymotryptic activity (Redfield & Anfinsen, 1956). After dialysis against water, the solution WB8 lyophilized and stored at -10°C. 21
316
J. D. STUBBS AND P. KAESBERG
Tryptic hydrolyses were carried out on both urea-denatured and heat-denatured BMV protein. Denaturation with urea was carried out essentially by the procedure of Helinski & Yanofsky (1962) on BMV protein that had been dialysed exhaustively against water. In a typical experiment, 90 mg of BMV protein were dried by rotary evaporation at 45°C under vacuum. The dried protein was dissolved in 9 ml, of 6 M-urea in 0·3 M-ammonium bicarbonate (pH 7,9). The protein solution was heated to 60°C and kept there for 20 min at which time 18 mI. of water were added to make the solution 2 M with respect to urea. Invariably it became densely turbid at this point. The temperature was reduced to 37°C and hydrolysis initiated by addition of 0·9 ml. of a 0·1 % solution of trypsin in water. The digestion mixture was kept dispersed by stirring. The pH changed less than 0·2 pH units during the digestion period (usually 4 hr), Digestion was terminated by adjusting the pH to 3·3 with glacial acetic acid. In some experiments the rate of digestion in the absence of ammonium bicarbonate was followed using an autotitrator with automatic recording of NaOH consumption required to maintain constant pH. The digestion mixture was centrifuged at 10,000 rev.fmin for 30 min in a Spinco model L, no. 40 rotor. The supernatant solution was decanted from the pelleted insoluble fraction, and the latter was dialysed against water and stored frozen. Urea was removed from the soluble peptides by absorbing them on an ion exchange column made of the cation exchange resin AG 50W-X2 (50-100 mesh, H+ form, Bio-Rad Oorp.), washing the column with water, and eluting the peptides with 4 N-NH 4 0 H . The eluate was taken to dryness by rotary evaporation and stored at - 10°C. In experiments involving heat-denatured protein, a 0·4% solution of BMV protein in water was stirred at 95°C for 30 min. The turbid suspension was rapidly cooled to 25°C and made 0·01 M with respect to ammonium bicarbonate. The suspension was warmed to 37°C and hydrolysis initiated by adding trypsin in water to give a final substrate to enzyme ratio of 50: 1 by weight. After 25 hr, the pH of the mixture was decreased to 6·0. The mixture was centrifuged at 10,000 rev.fmin for 30 min, the supernatant solution was decanted and stored at _10°C. The pelleted insoluble fraction was washed with water and stored frozen. (v) Fractionation of the tryptic peptides Separations of the peptides resulting from tryptic digestion were made by chromatography and electrophoresis on 18·25 in. x 22'5 in. sheets of Whatman no. 3 MM filter paper. One to two mg of the dried soluble fractions were taken up in 0·05 ml. of water and applied at the center of the long side of the paper, 3 in. below the top edge. The bottom edge was serrated to permit uniform flow of solvent from the paper. Chromatograms were run descendingly at 20°C for 22 hr in a closed glass tank. The solvent was freshly-prepared isoamyl alcohol-pyridine-water (35:35:30 by vol.) (Baglioni, 1961). In the second dimension, paper electrophoresis was carried out for 1 hr in pyridine-acetic acid-water (100: 4: 900, by vol., pH 6,4) with a Savant model LT36 electrophoresis apparatus (Savant Instruments, Inc., 221 Park Ave., Hicksville, N.Y.). The voltage was 2600 with currents drawn varying between 110 and 130 rnA. The peptides were located by dipping the dried paper in 0'5% ninhydrin in acetone and allowing development at room temperature. Alternatively, the chlorination-starch-iodine procedure was used, as modified by Schwartz & Pallansch (1958). Arginine-containing peptides were detected with a modified Sakaguchi reagent (Smith, 1953a). Tryptophancontaining peptides were located with the Ehrlich reagent (Smith, 1953b).
(vi) Column chromatography Peptides were separated on a preparative scale by column chromatography following a procedure similar to that reported by Konigsberg & Hill (1962). Chromatography was carried out at 40°C on 1 em x 150 em columns of the cation exchange resin AG 50W-X2 Aminex (blended 50% 120-200 mesh plus 50% 200-235 mesh ammonium form, Bio-Rad Corp.) that had been prewashed with NaOH, water, HCI, and water, respectively. Fifty to 100 mg of peptides resulting from tryptic digestion of urea-denatured protein were taken up in 3 ml, of 25% acetic acid and immediately applied to columns that had been equilibrated with pyridinium acetate buffer, pH 4·8 (14 ml. pyridine plus 15 ml. acetic acid per liter of aqueous solution). Gradient elution was carried out in which the elution
BROMEGRASS MOSAIC VIRUS PROTEIN
317
medium WIlB the above pyridinium acetate buffer, pH 4'8, linearly augmented with pyridinium acetate buffer, pH 5·4 (684 ml. pyridine plus 180 ml, acetic acid per liter of aqueous solution). Flow rates were adjusted to approximately 0·2 ml.jrnin with II. motordriven, controlled- volume pump (Milton Roy Co., 1300 E. Mermaid Lane, Philadelphia 18, Pa.). Fractions were collected automatically at 9 min intervals. The peptides were located by an assay divised by Satake, Okuyama, Ohashi & Shinoda (1960)88 modified by Yamazaki (personal communication). One-half ml. samples from each fraction were dried in a vacuum oven at 45°C. The dried fractions were dissolved in 2 ml. of 0'5 H-sodium bicarbonate in water. One ml. portions of a 0'1 % aqueous solution of 2,4,6.trinitrobenzene-l-sulfonic acid were added to each fraction and the tubes were kept for 2 hr at 37°C in the dark. The absorbency which developed at 420 mp. was then taken 88 a measure of peptide concentration.
3. Results (i) Degradation of BMV in 1 M-calcium chloride Figure 1 gives the u.v. absorption spectrum obtained from the supernatant liquid resulting from calcium chloride treatment of BMV. It has a maximum at 275 mp', a minimum at 249 uu», and an inflection point at 290 mp.. The ratio of the absorbancy 1·1 -
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at 260 mp. to that at 280 mp. is 0·62. The spectrum is typical of that expected for a. protein solution which is substantially free of nucleic acid and undegraded virus. From the known absorbency indicea ofBMV RNA (21·4 cm 2jmg at 260 uu» and 10·8 cm 2jmg at 280 mfL) and from those ofBMV protein (0·46 em2jmgand 0·76 em 2jmg, respectively, calculated from the content of tryptophan, tyrosine and phenylalanine), the amount
318
J. D. STUBBS AND P. KAESBERG
of RNA in the supernatant solution was less than 0·1 % of that of the protein. The supernatant liquid contained more than 95% of the viral protein. The effectiveness of the fractionation obtainable with this procedure has already been documented (Yamazaki & Kaesberg, 1963a). (ii) Sedimentation analyses
Ultracentrifugal analysis of the supernatant liquid showed a single, slowly sedimenting boundary with a base line that exhibited pronounced curvature. Because of the uncertainty involved in quantitative interpretation of analyses in high molarity salt solutions, it was desirable to dialyse the supernatant solution against more suitable ionic media. Dialysis against several changes of 0·1 M-sodium cacodylate (pH 6'5) at 5°C for times varying between 20 and 40 hours resulted in small amounts of a fibrous precipitate which was insoluble in 0·1 N -sodium hydroxide and iu 0·1 N -hydrochloric acid. Sedimentation of the clarified protein solution resulted in a single symmetric boundary, sgo,w = 2·8 s. Similar results were obtained in 0·1 M-tris-chloride (pH 6'1) and in 0·1 M-calcium chloride (pH 5,6). The sedimentation coefficients in these buffers were independent of concentration in the concentration range 0 to 0·9%. The protein was observed to aggregate at several pH values between 7·5 and 9. A solution dialysed for 12 hours against 0·1 M-tris-chloride (pH 7'5) exhibited considerable turbidity. This solution (after clarification by centrifugation) gave a. single sedimenting boundary (8 20,w = 2·8 s) with a pronounced leading edge. Some of the pelleted material from the clarification step could be dissolved in 0·1 M-sodium cacodylate (pH 6,0) and exhibited a single symmetric sedimentation peak, sgo w = 2·8 s. The protein was found to be quantitatively precipitated after four hours' dia'lysis against 0·1 M-sodium glycinate (pH 9). Only part of the precipitate was soluble on readjustment to pH 6,0, and this sedimented as a single component, Bgow = 2·8 s. The effect of pH lower than 5·6 was not studied in detail. (iii) Molecular weight Table 1 gives the results of approach to equilibrium sedimentation analyses of calcium chloride-treated protein, which had been dialysed exhaustively against 0·1 M-sodium cacodylate, pH 6·0. It may be seen that SID values did not decrease TABLE
1
Molecular weights of calcium chloride-prepared BM V protein in 0·1 M -sodium ca.rodylate, pH 6·0, as a function of time Minutes after reaching 19,160 rev.rmin 90 122 154 186
(SID
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101 see"/em)" 4·17 4·54 4·26 4·42
Molecular weight 38,000 41,400 38,900 40,300 Average = 39,700
with time. As has been shown by Ehrenberg (1957), constant or slightly increasing SID is evidence of homogeneity. It is concluded that the BMV protein under these conditions was homogeneous with respect to molecular weight. From Table 1 its
319
BROMEGRASS )lOSAIC VIRUS PROTEIN
molecular weight was 40 x 1()3 ± 2 x 1()3. Similar results were obtained when dialysis was against 0·1 M·tris-chloride (pH 6'5) and against 0·1 M-calcium chloride (pH 5'6). (iv) Amino acid analyses Amino acid analyses are presented in Table 2. Columns 2 through 5 give the mole percentages recovered in each analysis. The recoveries of serine, threonine and tyrosine were observed to decrease with time of hydrolysis. Their values, extrapolated TABLE
2
Amino acid composition of BMV protein
15
Lysine Histidine Arginine Aspartic aeid§ Threonine Serine Glutamic a.cid§ Proline Glycine Alanine Valine Methionine'] Isoleucine Leucine Tyrosine Phenylalanine
6·27 2·37 6·79 5·66 5·91 6·82 9·99 3'73 5·69 18·38 8·38 1·60 4·28 8'46 2·74 2·76
Hydrolysis time (hr) 24 38 6·41 2·20 7·24 5·64 5·80 6·47 10·02 3·86 5·54 18·41 8·80 1·49 4·43 8·35 2·65 2·70
6·53 1·78 7·31 5·68 5·69 6·16 9·87 3·83 5·59 18·31 9·30 1·58 4·52 8·42 2·47 2·74
Average 60 7·10 1'70 7·33 5·67 5·46 5·60 9·94 3·62 5·66 18'52 9·96 1'59 4·61 8'40 2·45 2·73
Cysteine~
Tryptophan t t
HOt 2·01 H 3t 5·66 6'0q 7· 15t 9·95 3·76 5·62 18·40 9·9 6t 1-56 4-61 t 8·41 2'77t 2·73 0·52 1·12
Relative Residues molar per ratio subunit 12·7 3·7 13-1 10·0 10·7 12·8 17·8 6·7 10·0 32·8 17·8 2·8 8·2 15·0 4'9 4·9 1·0 2·0
13 4 13 10 11 13 18 7 10 33 18 3 8 15 5 5 1 2
Total 189
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60-hour value,
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Extrapolated to zero hydrolysis time, § Combined sum of amide and acid,
II Detennined as methionine plus methionine sulfoxides, '\I Determined as cysteic acid with performic acid-oxidized protein, tt Determined from alkali spectrum of protein in 0·1 N·:-l"aOH.
to zero time, are recorded in the sixth column. The amounts of valine, isoleucine, lysine and arginine were observed to increase with hydrolysis time. The 60-hour values are recorded in the fifth column. The molar ratio of tyrosine to tryptophan, calculated from the u.v. spectrum of BMV protein in 0·1 N-sodium hydroxide, according to the method of Goodwin & Morton (1946), was found to be 2·5 to 1.
(v) Tryptic peptide maps
In the case of urea-denatured BMV protein, the turbidity which was observed upon the addition of urea was almost completely eliminated after 90 minutes of digestion with trypsin (at pH 7·9, 37°C). Digestion, as followed with an autotitrator, was about 80% complete in four hours. At the end of that time the digestion rate was low but still measurably greater than zero. After four hours' digestion, about one-fifth of the digestion mixture precipitated when the pH was lowered to 3·3. The properties of this precipitate have not been investigated extensively. However, its amino acid composition is different from that of
320
J. D. STUBBS AKD P. KAESBERG
BMV protein itself. Its proportions of valine, leucine, phenylalanine and tyrosine are relatively high. Its proportions of isoleucine arginine, and particularly lysine are low. We have not been able to bring any peptides from the precipitate into solution for possible study by electrophoresis or chromatography. Redigestion with trypsin for extended periods resulted in very little additional material soluble at pH 3·3. From the amino acid analyses above, we judge that the precipitating fraction contained several relatively large peptides with mostly non-polar amino acid residues and C-terminal arginine. Presumably there were also incompletely digested fragments and peptides occluded to the insoluble material. The fraction soluble at pH 3'3, doubtless contains most of the lysine- and argininecontaining tryptic peptides. A diagram of the peptide map obtained from it is presented in Fig. 2. There were 22 major ninhydrin spots with an additional four
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spots appearing as faint traces. The spot of origin exhibited a faint ninhydrin color. The 22 major spots were also major spots when detected by means of the chlorinationstarch-iodine stain while three trace ninhydrin spots appeared as trace spots. One trace ninhydrin spot was a major chlorination-starch-iodine spot. This spot may represent an Nvsubstituted terminal peptide such as is known to exist in the case of several other RNA-containing plant viruses. There were nine Sakaguchi-positive spots plus a trace of Sakaguchi-positive material at the origin. Two major ninhydrin spots were positive for the tryptophan-specific Ehrlich reagent. These results are summarized in Table 3. In the case of heat-denatured protein, the initial turbidity decreased slowly with digestion time, but considerable turbidity remained even after 25 hours' digestion. At the end of 25 hours, when the pH of the digest was lowered to 6,0, a precipitate formed which accounted for 35 to 40% of the original protein. This insoluble fraction could not be dissolved completely even at pH 2 and pH 12. Peptide maps from the soluble fraction were reminiscent of those from the urea- denatured soluble fraction, but, in general, resolution was poor.
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J. D. STUBBS Al\D P. KAESBERG
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(vi) Estimate of chemical subunit molecular weight The tryptic peptide map of the soluble fractions, above, evidently represented 23 major tryptic peptides, 22 of which terminated in lysine or arginine. Of these 22, 9 terminated with arginine and therefore 13 must have terminated with lysine. The corresponding insoluble fraction was low in lysine and we judge that it could account for no more than two additional lysine-containing tryptic peptides. Most probably it accounts for none. Thus, we conclude that the chemical subunit of BMV protein contains 13 to 15 lysine residues. From the amino acid analyses of BMV protein this corresponds to 189 to 221 amino acid residues and a subunit molecular weight between 20,300 and 25,700. Table 2, column 8 gives the numbers of amino acid residues in the subunit on the assumption of 13 lysine residues. TABLE
3
Major spots appearing on the tryptic peptide map of BMV protein
No, of peptides 9 11 2
Xinhydrin Sakaguchi
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Total 23
(vii) Partial separation of peptides on a preparative scale
Column chromatographic separation of the soluble peptides resulting from the tryptic digestion of urea-denatured protein is presented in Fig. 3. Absorbancy at 420 mfL after reaction with 2,4,6-trinitrobenzene-l-sulfonic acid is plotted versus volume. Seventeen major peaks were resolved. Preliminary correlations of these peaks with spots from the two-dimensional tryptic maps have been made. In some instances a column chromatography peak corresponded to a single spot and thus evidently represented a single peptide. In other eases a peak corresponded to two or more spots. It is our judgment that it will be possible, by means of column chromatography, to obtain many of the tryptic peptides in sufficient quantity and purity to warrant their detailed study.
4. Discussion The calcium ohloride procedure for preparing BMV protein is simple and gives a homogeneous soluble protein in high yield. Isolation of BMV protein by use of 67% acetic acid (Incardona, 1962) or by alkali (Stubbs, 1962) has been found to be less satisfactory. In both instances protein recovery is low and most of it is in the form of insoluble aggregates. The total number of tryptic peptides can serve as an indicator of the chemical molecular weight of a protein provided that lysine and arginine content are known, and if it may be assumed that trypsin cleaves specifically the peptide bonds involving the carboxyl groups of lysine and arginine. The reliability of the procedure does not depend on quantitative recovery but does depend on the ability to detect and count
BROMEGRASS :\lQSAIC VIRUS PROTEIN
323
the products of hydrolysis. We have determined from such data that the molecular weight of the chemical subunit of BMV protein is most likely 20,300 and is almost certainly in the range 20,000 to 25,000. The amino acid composition of the intact protein indicates a predominance of hydrophobic side-chains, a characteristic common to many other viruses. An unusual feature, however, is that over 17% of the residues are alanine. It is probable that hydrophobic bonds play an important role in the structural integrity of the virus. This may explain the higher susceptibility of urea-denatured protein than heatdenatured protein to tryptic hydrolysis. The protein isolated by the calcium chloride procedure has a molecular weight of about 40,000. Evidently it is a dimer of the chemical subunit. It is not entirely unexpected that the chemical subunit was not isolated in monomeric form. BMV, itself, is exceedingly soluble in aqueous media-about 0·7 saturated ammonium sulfate is required to precipitate it. That portion of its protein exposed to the solvent must thus have a high proportion of hydrophilic groups, the remaining portion must be quite hydrophobic. When the protein is converted to subunit form in the presence of calcium chloride, the hydrophobic regions evidently associate in such a way as to yield a dimer, If it may be assumed that the protein shell of BMV contains only a single species of protein, then this shell is constituted of 180 chemical subunits since the protein portion of BMV has a molecular weight of about 3·6 x 10 6 • We are indebted to Dr. Hiroshi Yamazaki for many helpful discussions. This work was supported by the U.S. Public Health Service. REFERENCES Anderegg, J. W., Wright, M. and Kaesberg, P. (1963). Biophsj«. J. 3,175. Baglioni, C. (1961). Biochim, biophys. Acta, 48, 392. Bockstahler, L. E. & Kaeaberg, P. (1962). Biophys. J. 2, 1. Ehrenberg, A. (1957). Acta Ohern. Scand, 11, 1257. Goodwin, T. W. & Morton, R. A. (1946). Biochem. J. 40, 628. Helinski, D. R. & Yanofsky, C. (1962). Biochim, biophys. Acta, 63, 10. Hire, C. H. W. (1956). J. Biol. Ohern. 219, 611. Incardona, N. (1962). Ph.D. Thesis, University of Wisconsin. Konigsberg, W. & Hill, R. J. (1962). J. Biol. Ohern. 237, 2547. Redfield, R. R. & Anfinsen, C. B. (1956). J. tu«. Ohern. 221, 385. Satake, K., Okuyama, T., Ohashi, M. & Shinoda, T. (1960). J. Biochem. 47, 654. Schwartz, D. P. & Pallansch, M. J. (1958). Analyt. Ohern. 30, 219. Smith, I. (1953a). Nature, 172, HOO. Smith. I. (1953b). Nature, 171, 43. Spackman, D. H., Stein, W. H. & Moore, S. (1958). Analyt. Ohern. 30, 1190. Stubbs, J. D. (1962). M.S. Thesis, University of Wisconsin. Yamazaki, H. & Kaesberg, P. (1963a). J. l"lol. Bwl. 7, 7GO. Yamazaki, H. & Kaesberg, P. (1963b). J. Mol. Biol. 6, 465.
A Protein Subunit of Bromegrass Mosaic Virus D. STUBBS AND PAUL KAESBERG Department of Biochemistry, University of Wisconsin Madison 6, Wisconsin, U.S.A. JOHN
(Received 11 October 1963) The protein and RNA of bromegrass mosaic virus were separated from each other by dialysis of the virus against 1 M-calcium chloride. In this medium the virus is degraded, the RNA is quantitatively precipitated and there remains a supernatant solution of viral protein. The protein obtained in this way was soluble in various buffers in the pH range 5·5 to 6·5 and exhibited a single symmetrical sedimenting boundary in the ultracentrifuge. Its molecular weight was 40,000 ± 2,000 as determined by the Ehrenberg method of approach to sedimentation equilibrium. Amino acid analyses indicated that there were about 52 lysyl plus arginyl residues per molecular weight of 40,000. Tryptic hydrolysis of ureadenatured protein yielded 23 soluble peptides and an insoluble residue amounting to about 20% by weight of the intact protein. From the number of soluble tryptic peptides and an assumed number of tryptic peptides in the insoluble residue it was concluded that the molecular weight of the protein subunit of bromegrass mosaic virus was slightly greater than 20,000 and that the protein moiety obtained by treatment with 1 M.CaCla was a relatively stable dimer of the subunit. Partial separation of the soluble peptides resulting from tryptic hydrolysis was achieved on a preparative scale by column chromatography.
1. Introduction BMVt is a small, nearly spherical, RNA-containing plant virus. Physical and chemical studies (Bockstahler & Kaesberg, 1962; Anderegg, Wright & Kaesberg, 1963) have shown that the virus particle contains 79% protein and 21% RNA. It weighs 4·6 x 10 6 atomic mass units. Its external diameter is about 260 A. It has a central cavity 80 A in diameter which is surrounded by a shell-shaped region containing the viral RNA, and this in turn is surrounded by a shell of structural protein, presumably in a symmetrical array of subunits. Yamazaki & Kaesberg (1963a) found that BMV is degraded in 1 M-calcium chloride to yield a soluble fraction which is exclusively protein and a precipitate which consists of the viral RNA. This simple procedure yields viral protein in a form suitable for further investigation. Broad bean mottle virus, which is superficially similar to BMV, is similarly degraded (Yamazaki & Kaesberg, 1963b), although in this case the precipitate contains appreciable protein. It is the purpose of this paper to present physical and chemical characteristics of the protein isolated by calcium chloride treatment of BMV.
t
Abbreviation used: BMV = bromegrass mosaic virus. 314
BROMEGRASS :\IOSAIC VIRUS PROTEIN
315
2. Materials and Methods (i) Preparation of BMF protein
BMV was isolated and purified as described by Bockstahler & Kaesberg (1962). The purified virus was treated with 1 M-CaCI2 in the following way. A suspension containing 1% BMV in water was dialysed for 24 hr at 5°C against 1 M-CaCI 2 plus 0·05 :\I-sodium cacodylate, pH 6·0. Approximately I 1. CaCI 2 solution was used for dialysis of 15 m!. of the BMV suspension contained in Visking 18/32 in. dialysis tubing. During dialysis, a flocculent white precipitate formed. The suspension was centrifuged at 10,000 rev./min for 30 min in a Spinco model L no. 40 rotor. The supernatant liquid, which contains the viral protein, was decanted from the pelleted precipitate, which contains the viral RNA, and was dialysed exhaustively against a suitable buffer or water. If such solutions were warmed above 10°C, considerable insoluble material formed. However, the solutions could be stored cold or frozen without loss of protein solubility or evidence of aggregation. Upon lyophilization, a considerable portion of the dried protein would not go back into solution under mild conditions. (ii) Analytical procedures Ultraviolet absorption spectra of BMV protein were obtained with a Cary Recording Spectrophotometer, model 11, equipped with a matched set of quartz cells of 1 cm path length. The spectra in the 230 to 290 mIL region were corrected for light scattering by extrapolation from the neighboring non-absorbing region. Ultracentrifuge analyses were made in the 12 mm synthetic boundary cell of a Spinco model E analytical ultracentrifuge equipped with a phase contrast Schlieren optical system. Sedimentation coefficicnts wcre determined at 59,780 rev.fmin at 4°C. In all runs an ionic strength of 0·1 or greater was maintained in order to swamp out charge effects. Sedimentation coefficients were corrected to standard conditions and are designated sgo,w' Molecular weights were determined by the Ehrenberg method of approach to equilibrium (Ehrenberg, 1957). In this procedure the ratio of the sedimentation coefficient to diffusion coefficient (8/ D) of a sedimenting substance is obtained from the sedimentation pattern near the meniscus of the solution as sedimentation equilibrium is approached. Such a determination involves two runs in one of which the protein boundary is made synthetically. In the experiments reported here, 0'40 m!. of an 0·8% solution of the protein exhaustively dialysod against an appropriate buffer were spun at 19,160 rcv.jmin in the 12 rom synthetic-boundary cell. Four exposures of the refractive index gradient curve were taken, the first, 90 min after reaching speed and the remaining three at 32 min intervals. Temperature was maintained at 1· 1°C. A second run was then made with the same cell and solution (redispersed by gentle shaking) but 0·30 m!. of equilibrated buffer were layered over the sample when the speed of rotation was 5000 to 7000 rev.lmui. In other details, the second run duplicated the first. Values of S/ D were obtained from these patterns and were inserted into the familiar Svedberg equation. The partial specific volume of the solute was taken as 0·74 m!./g (Bockstahler & Kaesbcrg, 1962). (iii) Amino acid anolsjsee For these analyses BMV protein in 1 M-CaCI 2 was dialysed exhaustively against distilled water. Approximately 4 mg of protein were dried and hydrolysed for various t.imes in 0'5 ml. of glass-distilled constant-boiling HCI at 110°C under nitrogen gas. The amino acid composition of the hydrolysates were determined on a Spinco model 120 automatic amino acid analyser as described by Spackman, Stein & Moore (1958). Cysteic acid was detennined on a protein sample oxidized with performic acid according to the method of Hirs (1956). Tryptophan was estimated from the ultraviolet absorption spectra of the protein in 0·1 N·NaOH according to the method of Goodwin & Morton (1946). (iv) Tryptic digestion Salt-free trypsin, twice crystallized (Worthington Biochemical Sales Corp.) was treated with 0·0625 N-HCI at 37°C for 18 hr to lower preferentially a possible contaminating chymotryptic activity (Redfield & Anfinsen, 1956). After dialysis against water, the solution WB8 lyophilized and stored at -10°C. 21
316
J. D. STUBBS AND P. KAESBERG
Tryptic hydrolyses were carried out on both urea-denatured and heat-denatured BMV protein. Denaturation with urea was carried out essentially by the procedure of Helinski & Yanofsky (1962) on BMV protein that had been dialysed exhaustively against water. In a typical experiment, 90 mg of BMV protein were dried by rotary evaporation at 45°C under vacuum. The dried protein was dissolved in 9 ml, of 6 M-urea in 0·3 M-ammonium bicarbonate (pH 7,9). The protein solution was heated to 60°C and kept there for 20 min at which time 18 mI. of water were added to make the solution 2 M with respect to urea. Invariably it became densely turbid at this point. The temperature was reduced to 37°C and hydrolysis initiated by addition of 0·9 ml. of a 0·1 % solution of trypsin in water. The digestion mixture was kept dispersed by stirring. The pH changed less than 0·2 pH units during the digestion period (usually 4 hr), Digestion was terminated by adjusting the pH to 3·3 with glacial acetic acid. In some experiments the rate of digestion in the absence of ammonium bicarbonate was followed using an autotitrator with automatic recording of NaOH consumption required to maintain constant pH. The digestion mixture was centrifuged at 10,000 rev.fmin for 30 min in a Spinco model L, no. 40 rotor. The supernatant solution was decanted from the pelleted insoluble fraction, and the latter was dialysed against water and stored frozen. Urea was removed from the soluble peptides by absorbing them on an ion exchange column made of the cation exchange resin AG 50W-X2 (50-100 mesh, H+ form, Bio-Rad Oorp.), washing the column with water, and eluting the peptides with 4 N-NH 4 0 H . The eluate was taken to dryness by rotary evaporation and stored at - 10°C. In experiments involving heat-denatured protein, a 0·4% solution of BMV protein in water was stirred at 95°C for 30 min. The turbid suspension was rapidly cooled to 25°C and made 0·01 M with respect to ammonium bicarbonate. The suspension was warmed to 37°C and hydrolysis initiated by adding trypsin in water to give a final substrate to enzyme ratio of 50: 1 by weight. After 25 hr, the pH of the mixture was decreased to 6·0. The mixture was centrifuged at 10,000 rev.fmin for 30 min, the supernatant solution was decanted and stored at _10°C. The pelleted insoluble fraction was washed with water and stored frozen. (v) Fractionation of the tryptic peptides Separations of the peptides resulting from tryptic digestion were made by chromatography and electrophoresis on 18·25 in. x 22'5 in. sheets of Whatman no. 3 MM filter paper. One to two mg of the dried soluble fractions were taken up in 0·05 ml. of water and applied at the center of the long side of the paper, 3 in. below the top edge. The bottom edge was serrated to permit uniform flow of solvent from the paper. Chromatograms were run descendingly at 20°C for 22 hr in a closed glass tank. The solvent was freshly-prepared isoamyl alcohol-pyridine-water (35:35:30 by vol.) (Baglioni, 1961). In the second dimension, paper electrophoresis was carried out for 1 hr in pyridine-acetic acid-water (100: 4: 900, by vol., pH 6,4) with a Savant model LT36 electrophoresis apparatus (Savant Instruments, Inc., 221 Park Ave., Hicksville, N.Y.). The voltage was 2600 with currents drawn varying between 110 and 130 rnA. The peptides were located by dipping the dried paper in 0'5% ninhydrin in acetone and allowing development at room temperature. Alternatively, the chlorination-starch-iodine procedure was used, as modified by Schwartz & Pallansch (1958). Arginine-containing peptides were detected with a modified Sakaguchi reagent (Smith, 1953a). Tryptophancontaining peptides were located with the Ehrlich reagent (Smith, 1953b).
(vi) Column chromatography Peptides were separated on a preparative scale by column chromatography following a procedure similar to that reported by Konigsberg & Hill (1962). Chromatography was carried out at 40°C on 1 em x 150 em columns of the cation exchange resin AG 50W-X2 Aminex (blended 50% 120-200 mesh plus 50% 200-235 mesh ammonium form, Bio-Rad Corp.) that had been prewashed with NaOH, water, HCI, and water, respectively. Fifty to 100 mg of peptides resulting from tryptic digestion of urea-denatured protein were taken up in 3 ml, of 25% acetic acid and immediately applied to columns that had been equilibrated with pyridinium acetate buffer, pH 4·8 (14 ml. pyridine plus 15 ml. acetic acid per liter of aqueous solution). Gradient elution was carried out in which the elution
BROMEGRASS MOSAIC VIRUS PROTEIN
317
medium WIlB the above pyridinium acetate buffer, pH 4'8, linearly augmented with pyridinium acetate buffer, pH 5·4 (684 ml. pyridine plus 180 ml, acetic acid per liter of aqueous solution). Flow rates were adjusted to approximately 0·2 ml.jrnin with II. motordriven, controlled- volume pump (Milton Roy Co., 1300 E. Mermaid Lane, Philadelphia 18, Pa.). Fractions were collected automatically at 9 min intervals. The peptides were located by an assay divised by Satake, Okuyama, Ohashi & Shinoda (1960)88 modified by Yamazaki (personal communication). One-half ml. samples from each fraction were dried in a vacuum oven at 45°C. The dried fractions were dissolved in 2 ml. of 0'5 H-sodium bicarbonate in water. One ml. portions of a 0'1 % aqueous solution of 2,4,6.trinitrobenzene-l-sulfonic acid were added to each fraction and the tubes were kept for 2 hr at 37°C in the dark. The absorbency which developed at 420 mp. was then taken 88 a measure of peptide concentration.
3. Results (i) Degradation of BMV in 1 M-calcium chloride Figure 1 gives the u.v. absorption spectrum obtained from the supernatant liquid resulting from calcium chloride treatment of BMV. It has a maximum at 275 mp', a minimum at 249 uu», and an inflection point at 290 mp.. The ratio of the absorbancy 1·1 -
/-', 0·9
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at 260 mp. to that at 280 mp. is 0·62. The spectrum is typical of that expected for a. protein solution which is substantially free of nucleic acid and undegraded virus. From the known absorbency indicea ofBMV RNA (21·4 cm 2jmg at 260 uu» and 10·8 cm 2jmg at 280 mfL) and from those ofBMV protein (0·46 em2jmgand 0·76 em 2jmg, respectively, calculated from the content of tryptophan, tyrosine and phenylalanine), the amount
318
J. D. STUBBS AND P. KAESBERG
of RNA in the supernatant solution was less than 0·1 % of that of the protein. The supernatant liquid contained more than 95% of the viral protein. The effectiveness of the fractionation obtainable with this procedure has already been documented (Yamazaki & Kaesberg, 1963a). (ii) Sedimentation analyses
Ultracentrifugal analysis of the supernatant liquid showed a single, slowly sedimenting boundary with a base line that exhibited pronounced curvature. Because of the uncertainty involved in quantitative interpretation of analyses in high molarity salt solutions, it was desirable to dialyse the supernatant solution against more suitable ionic media. Dialysis against several changes of 0·1 M-sodium cacodylate (pH 6'5) at 5°C for times varying between 20 and 40 hours resulted in small amounts of a fibrous precipitate which was insoluble in 0·1 N -sodium hydroxide and iu 0·1 N -hydrochloric acid. Sedimentation of the clarified protein solution resulted in a single symmetric boundary, sgo,w = 2·8 s. Similar results were obtained in 0·1 M-tris-chloride (pH 6'1) and in 0·1 M-calcium chloride (pH 5,6). The sedimentation coefficients in these buffers were independent of concentration in the concentration range 0 to 0·9%. The protein was observed to aggregate at several pH values between 7·5 and 9. A solution dialysed for 12 hours against 0·1 M-tris-chloride (pH 7'5) exhibited considerable turbidity. This solution (after clarification by centrifugation) gave a. single sedimenting boundary (8 20,w = 2·8 s) with a pronounced leading edge. Some of the pelleted material from the clarification step could be dissolved in 0·1 M-sodium cacodylate (pH 6,0) and exhibited a single symmetric sedimentation peak, sgo w = 2·8 s. The protein was found to be quantitatively precipitated after four hours' dia'lysis against 0·1 M-sodium glycinate (pH 9). Only part of the precipitate was soluble on readjustment to pH 6,0, and this sedimented as a single component, Bgow = 2·8 s. The effect of pH lower than 5·6 was not studied in detail. (iii) Molecular weight Table 1 gives the results of approach to equilibrium sedimentation analyses of calcium chloride-treated protein, which had been dialysed exhaustively against 0·1 M-sodium cacodylate, pH 6·0. It may be seen that SID values did not decrease TABLE
1
Molecular weights of calcium chloride-prepared BM V protein in 0·1 M -sodium ca.rodylate, pH 6·0, as a function of time Minutes after reaching 19,160 rev.rmin 90 122 154 186
(SID
X
101 see"/em)" 4·17 4·54 4·26 4·42
Molecular weight 38,000 41,400 38,900 40,300 Average = 39,700
with time. As has been shown by Ehrenberg (1957), constant or slightly increasing SID is evidence of homogeneity. It is concluded that the BMV protein under these conditions was homogeneous with respect to molecular weight. From Table 1 its
319
BROMEGRASS )lOSAIC VIRUS PROTEIN
molecular weight was 40 x 1()3 ± 2 x 1()3. Similar results were obtained when dialysis was against 0·1 M·tris-chloride (pH 6'5) and against 0·1 M-calcium chloride (pH 5'6). (iv) Amino acid analyses Amino acid analyses are presented in Table 2. Columns 2 through 5 give the mole percentages recovered in each analysis. The recoveries of serine, threonine and tyrosine were observed to decrease with time of hydrolysis. Their values, extrapolated TABLE
2
Amino acid composition of BMV protein
15
Lysine Histidine Arginine Aspartic aeid§ Threonine Serine Glutamic a.cid§ Proline Glycine Alanine Valine Methionine'] Isoleucine Leucine Tyrosine Phenylalanine
6·27 2·37 6·79 5·66 5·91 6·82 9·99 3'73 5·69 18·38 8·38 1·60 4·28 8'46 2·74 2·76
Hydrolysis time (hr) 24 38 6·41 2·20 7·24 5·64 5·80 6·47 10·02 3·86 5·54 18·41 8·80 1·49 4·43 8·35 2·65 2·70
6·53 1·78 7·31 5·68 5·69 6·16 9·87 3·83 5·59 18·31 9·30 1·58 4·52 8·42 2·47 2·74
Average 60 7·10 1'70 7·33 5·67 5·46 5·60 9·94 3·62 5·66 18'52 9·96 1'59 4·61 8'40 2·45 2·73
Cysteine~
Tryptophan t t
HOt 2·01 H 3t 5·66 6'0q 7· 15t 9·95 3·76 5·62 18·40 9·9 6t 1-56 4-61 t 8·41 2'77t 2·73 0·52 1·12
Relative Residues molar per ratio subunit 12·7 3·7 13-1 10·0 10·7 12·8 17·8 6·7 10·0 32·8 17·8 2·8 8·2 15·0 4'9 4·9 1·0 2·0
13 4 13 10 11 13 18 7 10 33 18 3 8 15 5 5 1 2
Total 189
t
60-hour value,
t
Extrapolated to zero hydrolysis time, § Combined sum of amide and acid,
II Detennined as methionine plus methionine sulfoxides, '\I Determined as cysteic acid with performic acid-oxidized protein, tt Determined from alkali spectrum of protein in 0·1 N·:-l"aOH.
to zero time, are recorded in the sixth column. The amounts of valine, isoleucine, lysine and arginine were observed to increase with hydrolysis time. The 60-hour values are recorded in the fifth column. The molar ratio of tyrosine to tryptophan, calculated from the u.v. spectrum of BMV protein in 0·1 N-sodium hydroxide, according to the method of Goodwin & Morton (1946), was found to be 2·5 to 1.
(v) Tryptic peptide maps
In the case of urea-denatured BMV protein, the turbidity which was observed upon the addition of urea was almost completely eliminated after 90 minutes of digestion with trypsin (at pH 7·9, 37°C). Digestion, as followed with an autotitrator, was about 80% complete in four hours. At the end of that time the digestion rate was low but still measurably greater than zero. After four hours' digestion, about one-fifth of the digestion mixture precipitated when the pH was lowered to 3·3. The properties of this precipitate have not been investigated extensively. However, its amino acid composition is different from that of
320
J. D. STUBBS AKD P. KAESBERG
BMV protein itself. Its proportions of valine, leucine, phenylalanine and tyrosine are relatively high. Its proportions of isoleucine arginine, and particularly lysine are low. We have not been able to bring any peptides from the precipitate into solution for possible study by electrophoresis or chromatography. Redigestion with trypsin for extended periods resulted in very little additional material soluble at pH 3·3. From the amino acid analyses above, we judge that the precipitating fraction contained several relatively large peptides with mostly non-polar amino acid residues and C-terminal arginine. Presumably there were also incompletely digested fragments and peptides occluded to the insoluble material. The fraction soluble at pH 3'3, doubtless contains most of the lysine- and argininecontaining tryptic peptides. A diagram of the peptide map obtained from it is presented in Fig. 2. There were 22 major ninhydrin spots with an additional four
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spots appearing as faint traces. The spot of origin exhibited a faint ninhydrin color. The 22 major spots were also major spots when detected by means of the chlorinationstarch-iodine stain while three trace ninhydrin spots appeared as trace spots. One trace ninhydrin spot was a major chlorination-starch-iodine spot. This spot may represent an Nvsubstituted terminal peptide such as is known to exist in the case of several other RNA-containing plant viruses. There were nine Sakaguchi-positive spots plus a trace of Sakaguchi-positive material at the origin. Two major ninhydrin spots were positive for the tryptophan-specific Ehrlich reagent. These results are summarized in Table 3. In the case of heat-denatured protein, the initial turbidity decreased slowly with digestion time, but considerable turbidity remained even after 25 hours' digestion. At the end of 25 hours, when the pH of the digest was lowered to 6,0, a precipitate formed which accounted for 35 to 40% of the original protein. This insoluble fraction could not be dissolved completely even at pH 2 and pH 12. Peptide maps from the soluble fraction were reminiscent of those from the urea- denatured soluble fraction, but, in general, resolution was poor.
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J. D. STUBBS Al\D P. KAESBERG
322
(vi) Estimate of chemical subunit molecular weight The tryptic peptide map of the soluble fractions, above, evidently represented 23 major tryptic peptides, 22 of which terminated in lysine or arginine. Of these 22, 9 terminated with arginine and therefore 13 must have terminated with lysine. The corresponding insoluble fraction was low in lysine and we judge that it could account for no more than two additional lysine-containing tryptic peptides. Most probably it accounts for none. Thus, we conclude that the chemical subunit of BMV protein contains 13 to 15 lysine residues. From the amino acid analyses of BMV protein this corresponds to 189 to 221 amino acid residues and a subunit molecular weight between 20,300 and 25,700. Table 2, column 8 gives the numbers of amino acid residues in the subunit on the assumption of 13 lysine residues. TABLE
3
Major spots appearing on the tryptic peptide map of BMV protein
No, of peptides 9 11 2
Xinhydrin Sakaguchi
+ + +
+
Chlorinationstarchiodine
+ + + +
Ehrlich
+
Total 23
(vii) Partial separation of peptides on a preparative scale
Column chromatographic separation of the soluble peptides resulting from the tryptic digestion of urea-denatured protein is presented in Fig. 3. Absorbancy at 420 mfL after reaction with 2,4,6-trinitrobenzene-l-sulfonic acid is plotted versus volume. Seventeen major peaks were resolved. Preliminary correlations of these peaks with spots from the two-dimensional tryptic maps have been made. In some instances a column chromatography peak corresponded to a single spot and thus evidently represented a single peptide. In other eases a peak corresponded to two or more spots. It is our judgment that it will be possible, by means of column chromatography, to obtain many of the tryptic peptides in sufficient quantity and purity to warrant their detailed study.
4. Discussion The calcium ohloride procedure for preparing BMV protein is simple and gives a homogeneous soluble protein in high yield. Isolation of BMV protein by use of 67% acetic acid (Incardona, 1962) or by alkali (Stubbs, 1962) has been found to be less satisfactory. In both instances protein recovery is low and most of it is in the form of insoluble aggregates. The total number of tryptic peptides can serve as an indicator of the chemical molecular weight of a protein provided that lysine and arginine content are known, and if it may be assumed that trypsin cleaves specifically the peptide bonds involving the carboxyl groups of lysine and arginine. The reliability of the procedure does not depend on quantitative recovery but does depend on the ability to detect and count
BROMEGRASS :\lQSAIC VIRUS PROTEIN
323
the products of hydrolysis. We have determined from such data that the molecular weight of the chemical subunit of BMV protein is most likely 20,300 and is almost certainly in the range 20,000 to 25,000. The amino acid composition of the intact protein indicates a predominance of hydrophobic side-chains, a characteristic common to many other viruses. An unusual feature, however, is that over 17% of the residues are alanine. It is probable that hydrophobic bonds play an important role in the structural integrity of the virus. This may explain the higher susceptibility of urea-denatured protein than heatdenatured protein to tryptic hydrolysis. The protein isolated by the calcium chloride procedure has a molecular weight of about 40,000. Evidently it is a dimer of the chemical subunit. It is not entirely unexpected that the chemical subunit was not isolated in monomeric form. BMV, itself, is exceedingly soluble in aqueous media-about 0·7 saturated ammonium sulfate is required to precipitate it. That portion of its protein exposed to the solvent must thus have a high proportion of hydrophilic groups, the remaining portion must be quite hydrophobic. When the protein is converted to subunit form in the presence of calcium chloride, the hydrophobic regions evidently associate in such a way as to yield a dimer, If it may be assumed that the protein shell of BMV contains only a single species of protein, then this shell is constituted of 180 chemical subunits since the protein portion of BMV has a molecular weight of about 3·6 x 10 6 • We are indebted to Dr. Hiroshi Yamazaki for many helpful discussions. This work was supported by the U.S. Public Health Service. REFERENCES Anderegg, J. W., Wright, M. and Kaesberg, P. (1963). Biophsj«. J. 3,175. Baglioni, C. (1961). Biochim, biophys. Acta, 48, 392. Bockstahler, L. E. & Kaeaberg, P. (1962). Biophys. J. 2, 1. Ehrenberg, A. (1957). Acta Ohern. Scand, 11, 1257. Goodwin, T. W. & Morton, R. A. (1946). Biochem. J. 40, 628. Helinski, D. R. & Yanofsky, C. (1962). Biochim, biophys. Acta, 63, 10. Hire, C. H. W. (1956). J. Biol. Ohern. 219, 611. Incardona, N. (1962). Ph.D. Thesis, University of Wisconsin. Konigsberg, W. & Hill, R. J. (1962). J. Biol. Ohern. 237, 2547. Redfield, R. R. & Anfinsen, C. B. (1956). J. tu«. Ohern. 221, 385. Satake, K., Okuyama, T., Ohashi, M. & Shinoda, T. (1960). J. Biochem. 47, 654. Schwartz, D. P. & Pallansch, M. J. (1958). Analyt. Ohern. 30, 219. Smith, I. (1953a). Nature, 172, HOO. Smith. I. (1953b). Nature, 171, 43. Spackman, D. H., Stein, W. H. & Moore, S. (1958). Analyt. Ohern. 30, 1190. Stubbs, J. D. (1962). M.S. Thesis, University of Wisconsin. Yamazaki, H. & Kaesberg, P. (1963a). J. l"lol. Bwl. 7, 7GO. Yamazaki, H. & Kaesberg, P. (1963b). J. Mol. Biol. 6, 465.