Characterization of avian leucosis group-specific antigen from avian myeloblastosis virus

388

BIOCHIMICAET BIOPHYSICAACTA

BBA 35193 C H A R A C T E R I Z A T I O N OF AVIAN LEUCOSIS G R O U P - S P E C I F I C A N T I G E N FROM AVIAN MYELOBLASTOSIS VIRUS

D A V I D W. A L L E N

The John Collins Warren Laboratories of the Huntington Memorial Hospital of Harvard University at the Massachusetts General Hospital, Boslon, Mass. (U.S.A .) (Received October 3oth, 1967)

SUMMARY

I. Avian leucosis group-specific antigen protein has been purified from avian myeloblastosis virus and studied b y methods suited for the small amounts available (0.2-0.5 rag). 2. Molecular weight determinations using the high speed equilibrium method demonstrate a weight average molecular weight of approx. 23 ooo, homogeneity of molecular size, and lack of concentration effect on molecular weight. 3. Amino acid analysis was performed with the use of an automatic amino acid analyzer. The number of dicarboxylic amino acid residues exceeds the number of dibasic amino acid residues. Since the protein is positively charged at p H 8.6 one would expect that some of the former are aminated. Proline is present in high concentration. 4- N o N-terminal amino acid could be detected using I14C!fluorodinitrobenzene even in the presence of 6 M guanidine hydrochloride or with special conditions of hydrolysis. It is suggested that the N-terminus is blocked. 5. Carboxypeptidase A releases alanine rapidly in amounts approximately equimolar to the amount of protein taken. Alanine seems a likely candidate for the C-terminal amino acid. 6. About 27 peptides were apparent after trypsin treatment. There are approx. 22 residues of arginine plus lysine in the protein, and one would predict 23 such peptides if the amino acids were arranged in a unique sequence. 7. Trypsin pretreatment of the group-specific antigen prevents the complement fixation reaction, implying that structural integrity of this protein is required for its antigenicity. 8. I t is concluded that group-specific antigen is a suitable protein for further investigation because of its interesting properties, and because it m a y represent a common gene in an important group of RNA oncogenic viruses.

Abbreviations: AMV, avian myeloblastosis virus; D N P , dinitrophenyl; F D N B , fluorodinitrobenzene. " This is publication No. 13 t 5 of the Cancer Colnmission of H a r v a r d University.

Biochim. Bioph3,s. ,4eta, 154 (1968) 388--396

AVIAN LEUCOSIS GROUP-SPECIFIC ANTIGEN

389

INTRODUCTION

Avian leucosis group-specific antigen 1, a common internal constituent of this class of viruses 2-4, has been isolated from avian myeloblastosis virus (AMV) by electrophoresis~. In this laboratory this antigen was purified by cellulose acetate electrophoresis and was found to be a protein free of RNA and host cell antigens, and to be homogeneous when re-electrophoresed on polyacrylamide gel 6. Even though less than milligram amounts of this protein are available, attempts at further characterization are being made because of its interest as a genetic product of oncogenic RNA viruses. Initial investigations of the molecular weight of this protein, its amino acid content, amino and carboxyl terminal amino acids, and tryptic peptides are reported in this paper.

METHODS

AMV was obtained from the blood of leukemic chicks largely following the methods of BEARD and co-workers ~. The virus was purified by differential centrifugation and solubilized using the Tween-ether method of ECKERT, ROTT AND SCH~FER 8, and the group-specific antigen was isolated by cellulose acetate electrophoresis (pH 8.6) as previously described 6. Group-specific antigen eluted from appropriate sections of the electrophoresis strips was pooled and concentrated, either by ultrafiltration or by dialysis and lyophilization. The homogeneity of each preparation was checked by polyacrylamide gel electrophoresis (pH 4.3) (ref. 6). Complement fixation assays were carried out essentially as described before 9. Rabbit sera are now prepared against purified group-specific antigen rather than against the cruder mixture of solubilized AMV proteins. Since these antisera do not fix complement with normal chicken tissue, absorption with this material is not required prior to use. Protein was measured by the method of LOWRY et al. 1°, using bovine serum albumin as a routine standard, with an appropriate correction based on a solution of weighed air-dried group-specific antigen. The Spinco Model E analytical ultracentrifuge was used for sedimentation equilibrium experiments, employing the Rayleigh interference optical system of the instrument and the high speed equilibrium method n. The group-specific antigen (I mg/ml) was dissolved in o.I-M phosphate buffer (pH 7.4) and centrifuged at 6-1o °. Photographs (Eastman, spectroscopic plates, Type IIG) were taken at frequent intervals to demonstrate conditions for sedimentation diffusion equilibrium. Interference patterns were measured with a Nikon Model 6 microcomparator, determining fringe displacement at o.I-mm intervals. Weight average and number average molecular weights were calculated as described by REES AND YOUNG12. Quantitative determination of amino acids was carried out using a Beckman Spinco Model 12o automatic amino acid analyzer is. To determine the amino acid composition, the group-specific protein was dissolved in I ml glass-distilled (approx. 6 M) HC1 in glass ampoules, O 2 removed with N2, the contents frozen with liquid N2, the ampoules evacuated, sealed, and the protein hydrolyzed at lO8 ° for various times (see RESULTS). To determine tyrosine and tryptophan, 118/,g'of group-specific protein t3iochim. Biophys. Acta, 154 (1908) 388 396

39 °

D.w. ALLEN

were dissolved in 0. 5 ,nl o.I M NaOH and the method of GOODWIN AND MORTON14 applied, using a Zeiss PMQ I I spectrophotometer. Hexosamine was determined by the pyrrole method, and the ultramicrotnodification of the Elson-Morgan procedure, as described by EXLEY 15. For this purpose 118 #g group-specific protein were hydrolyzed under milder conditions (4 M HC1, I00 °, 4 h). N- and C-terminal amino acids were determined as their I14C~dinitrophenyl (DNP) derivatives16,17./3-Alanine was used as an internal standard. The amino acids or proteins were shaken (2 h, 2o ~') in o.2 ml distilled water, with I/,mole (IO #C//~mole) of I~4C fluorodinitrobenzene (FDNB) (Nuclear-Chicago) in o.I ml ethanol with excess solid NaHCO a. After the alkaline reaction mixture was extracted 3 times with ether to remove the unreacted FDNB, and these extracts discarded, the mixture was acidified, and 2o-#g amounts of I~C IDNP amino acids (Mann Research Laboratories) in ~-butanol were added as carrier. D N P proteins were hydrolyzed under various conditions (see RESULTS). The ether and acid-water soluble D N P amino acids were obtained and the D N P amino acids were separated by 2-dimensional thin-layer chromatography using silica gel as recommended by BI~E~NE~, NIEDERWIESER AND PATAKIis. Radioactive spots on the chromatograms were located by autoradiography (4 days, Kodak NS 54 T X-ray fihn) and the [I~C!DNP amino acids coinciding with visible carrier D N P amino acids were eluted by scraping the silica into o.2 ml of o.I M HC1 and extracting the suspension 4 times with o.2 ml n-butanol-ethyl acetate (I :I, v/v). The combined extracts were brought up to m a r k in I-ml volumetric flasks with the same solvent, o.5-tnl aliquots were dried prior to measuring the radioactivity in an end window, low background, coincidence counter (Nuclear-Chicago). The amount of each carrier DNP amino acid recovered was determined spectrophotometrically, and factors were derived to calculate the recovery. Parallel experiments were done using an amino acid calibration mixture (Beckman Spinco Type I), diluted so that 0. 5 m/~mole of each amino acid was taken, and recovery factors for each amino acid relative to ~-alanine derived for a final correction procedure. Carboxypeptidase A, pretreated with diisopropylfluorophosphate (Worthington) was used for determining the C-terminal group 1~. Eighty two hundredths #g (0.024 re#mole) of this enzyme was added to 25/~g (I.O7 re#mole) of group-specific antigen in IO #1 0.025 M phosphate buffer. After various times of incubation (pH 7.4, 2o°), the reaction was stopped by the addition of 2 #1 of I M HC1, and the mixture was allowed to stand (o °, IO min). The o time control was run by adding HC1 prior to the carboxypeptidase. A further control was performed in which the carboxypeptidase was incubated without the group-specific antigen. Analyses of the amino acids released after various times of exposure to the enzyme utilized ~laC]FDNB as described above. Tryptic peptide mapping was performed by combining electrophoresis and chromatography on thin-layer chromatographic plates as described by STEGEMANN AND LEI~CH~°. 500 #g antigen, 5 #g trypsin (Worthington), and I/~1 of o.o5% phenol red were dissolved in 0.2 ml of o.I M ammonium carbonate buffer and incubated (4 h, 37 °, pH 7.8). The digest was lyophilized and placed in a vacuum desiccator containing anhydrous NaOH and concentrated H2GO 4 for 3-5 days, to remove the ammonium carbonate, then dissolved in Io #1 of water 4 #1 in I #1 portions were placed in one spot on a 20 cm × 2o cm cellulose plate (Macherey, Nagel and Co.) and electrophoresis carried out in pyridine acetic acid-water (3:12:485, v/v/v) (800 V, and run at Biochim. Bioph3,s. Acta 154 (1968) 388 396

AVIAN LEUCOSIS GROUP-SPECIFIC ANTIGEN

391

15 °, pH 3.9 for I h). Then the plate was dried in a stream of air, chromatographed in the second dimension with n-butanol-acetic acid-water (4:1:i, v/v/v), dried, rechromatographed in the same direction, and dried. Finally, the peptide map ~vas sprayed with o.2% ninhydrin in acetone, allowed to develop overnight in the dark, and the results recorded by photography. RESULTS

Molecular weight A preliminary determination at several speeds, and subsequent repetitions of the equilibrium sedimentation at 34 ooo rev./min gave consistent values of the molecular weight. A plot of the log of the concentration versus the square of the radial coordinate (Fig. I) provides evidence for homogeneity of particle size. Graphing the reciprocal of the number average molecular weight against concentration (Fig. 2) indicates lack

&3 3.1 2.9 :~2.7

~ 2.5

•

• •g;"

•

•

~

•

~ __

~2.3 2J 1.9 0

47,0

47"5

48.0

48.5

49.0

0.2

0.4

0.6

0.8

1.0

1.2

1,4

Concentration (ram fringe displacement)

~.6

Fig. I. Determination of ~veight average molecular weight by equilibrium sedimentation. Logarithm of concentration (mm fringe displacement) is plotted against the square of the distance in cm from the center of rotation. Fig. 2. Plot of the reciprocal of the number average molecular weight against concentration (ram fringe displacement).

of concentration effect on molecular weight. The specific volume calculated frmn the amino acid composition was 0.74 (ref. 21). Three determinations of the weight average molecular weight were 22 000, 25 000, and 23 ooo (average-_about 23 ooo). Amino acid analysis Amino acid analysis by the method of SPACKMAN,STEIN AND MOORE13 is shown in Table I. The first 3 analyses were performed on I5.8-/~g aliquots of group-specific antigen hydrolyzed for 20 h, 4 ° h and 7 ° h (lO8 °, 6 M HC1). A decrease in the content of serine, threonine, and tyrosine is observed with prolonged hydrolysis, as has been previously reported 2~. The 4th column represents an analysis of 34 °/~g of another preparation of group-specific antigen which had been oxidized 16 h at o ° with performic acid, then diluted with IO vol. of distilled water, lyophilized and hydrolyzed (20 h, 6 M HC1, lO8°). 4Ol/zg of amino acids were recovered, which, after subtracting the Biochim. Biophys. Acta, 154 (1968) 388-396

392

~. w. ALLEN

water added to the peptide bonds on hydrolysis, yields 342 #g of residues. A small peak possibly representing cysteic acid was present in the oxidized sample but this was also present in the unoxidized samples. Thus amounts of cysteine of the order of i residue per mole are possible but not proven 2~. The last column presents an estimate of the number of residues of each amino acid per mole of group-specific protein with, in most cases, an uncertainty of I or 2 residues. The results of the oxidized group-specific antigen were used in this calculation in view of the larger amounts analyzed, except for serine and threonine in which values TABLE

I

AMINO ACID COMPOSITION OF G R O U P - S P E C I F I C A N T I G E N

Residues/xoo residues recovered . . . . . . 2o h 4° h 7° h Oxidized

Residues amino acid per mole group-specific antigen

group-

specific antigen

(~o t~) Aspartic acid Threonine Serine Proline Glutanlic acid Glycine Alanine Methionine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan (by ultraviolet absorption)

7.9 7.3 5 .8 lO.2 lO.8 9.4 14.1 o 6.2 5 .o lO. 7 0. 7 i .8 3.1 0. 5 6. 5

8.o 6.6 5.5 lO. 3 li.i 9.7 14.1 o

8. ~ 6.8 3 .o I 1.6 11. 3 8.9 14.9 o

9.o 6.9 3 .6 i t.o Ii. 9 8.3 14.6 o

19 16 lZ 24 26 18 31

0,I

5.8

6.8

15

5 .2 lO.2 0. 5 ~ .5 4-2

5.4 lO. 7 0. 4 1.8 3.9

5-5 lO. 3 1. 4 3. z

12 22 2 3 7

0.8

0. 7

0. 5

I

6.2

6.(~

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15

2

of the 20 h unoxidized hydrolysate were taken. The tyrosine and tryptophan were determined by the spectrophotometric method 1~. The weight of amino acids recovered by analysis agrees approximately with the amount of group-specific antigen taken. Thus the group-specific antigen is largely protein, although small amounts of carbohydrate cannot be excluded. Micro methods for determination of hexosamine were set up 15, which in preliminary experiments yielded the expected hexosamine content from ovalbumin, and the presence of hexosamine in total AMV proteins was confirmed 24. However, no hexosamine was found in the group-specific antigen, nor in the solubilized AMV proteins it was derived from. In the T~veen-ether procedure of ECKERT, ROTT AND SCHXFER8 the hexosamine was confined to the proteins of the ether-phosphate buffer interface. The presence of small amounts of other carbohydrates in group-specific antigen has not been excluded. Biochim. Biophys. Acta, 154 (1968) 3 8 8 - 3 9 6

AVIAN LEUCOSIS GROUP-SPECIFIC ANTIGEN

393

N-terminal amino acid Five attempts have been made to determine the N-terminal amino acid group of the group-specific antigen, using [~4C!FDNB and IO m/~moles of protein each time. In preliminary experiments with similar amounts of known proteins, the expected N-terminal groups were obtained with rabbit hemoglobin (purified by electrophoresis), bovine serum albumin (Pentex), and bovine pancreatic ribonuclease (Worthington). With group-specific antigen no a D N P amino acid could be identified in the usual hydrolysate: (6 M HCI, 16 h, lO8°). Nor was there an a D N P amino acid when the reaction was carried out in 6 M guanidine hydrochloride (LIN, TSUNG AND ~'RAENKELCONRAT25). In other experiments the hydrolysis of the D N P group-specific protein was modified to preserve a possible N-terminal DNP-proline, or DNP-glycine, but neither amino acid appeared as the N-terminal group 2~. In no case were possible N-terminal DNP-amino acids obtained in more than i o % of the expected yield of one residue per molecule. On the other hand, amounts of the non-N-terminal epsilon DNP-lysine and 0-DNP-ty~'osine were found, consistent with their content in group-specific antigen. These observations indicate that the N-terminal group m a y be blocked. C-terminal amino acids The carboxyl terminal amino acid was investigated, using carboxypeptidase A and analyzing the released amino acids with [14C!FDNB. Fig. 3 shows the results of an analysis of the amino acids released from I re#mole group-specific antigen by carboxypeptidase A in 15 rain. On the left is a photograph of the 2-dimensional chromatogram in which the carrier D N P amino acids are visible and on the right the corresponding autoradiogram with tracings of the D N P amino acids. It is evident that the only amino acid released to a significant extent in this time period was alanine. Other small amounts of radioactivity did not correspond with D N P amino acids, were

Fig. 3. Amino acids released f r o m group-specific protein b y c a r b o x y p e p t i d a s e A in 1.5 min a f t e r reaction with [~4C]FDNB, addition of [~2C]DNP amino acids as carrier, and 2-dimensional t h i n layer c h r o m a t o g r a p h y , fl-Alanine was added to the enzyme digest in a m o u n t s equivalent to the group-specific protein as an internal standard. On the left is a p h o t o g r a p h of the carrier DNI" amino acids. On the right is an a u t o r a d i o g r a p h showing t h a t in addition to the/~-alanine internal s t a n d a r d , the only a m i n o acid p r e s e n t to react with the [x4C~.FDNB was alanine. The s t a n d a r d a b b r e v i a t i o n s for amino acids in this figure refer to their D N P derivatives.

Biochim. Biophys. Acta, 154 (1968) 388-396

394

1). W. ALLEN

1,6

1.4 ~ t2 ~ 1.0 ~0~ ca. ~ a~ .~ 0A

g

g o.2

i ,

~

;

4

5

Time (h)

6

7

8

Fig. 4. Time course of release of various amino acids from group-specific antigen by carboxypeptidase A. Alanine, ~; leucine and isoleucine, + ; serine, L?; threonine. @; glycine, ~; phenylalanine, ~ ; proline, ~ ; valine, ~ ; aspartic acid, @; glutamic acid, ~ ; tryptophan, [~.

present a o time, and did not increase with time. No alanine was present in the o time control or carboxypeptidase-alone control. The alanine continued to increase with incubation until its radioactivity equaled the fi-alanine, which had been added as an internal standard and was equivalent to the number of moles of group-specific antigen taken. Fig. 4 shows the average of 3 experiments with several time points, in which the amount of radioactivity in the eluted DNP amino acids was determined, and the amount of amino acids estimated, as described under MEWHODS. It is seen that the alanine increased rapidly and reached a value approximately equivalent to the moles of group-specific protein taken. The only other amino acids of possible significance were leucine and/or isoleucine, which were released at a slower initial rate, and may represent the next amino acid(s) in from the carboxyl terminus.

Tryptic peptide mapping A photograph of a tryptic peptide map of group-specific antigen is shown in Fig. 5. It is evident that 27 peptides can be identified. The combined lysine and

front o phenol

,e,

8O

,o, 0

,o

°"

0,,

20

q (+)

0

2

"origin

,5

6(3 0 '2 ~
5<~ 4

f~ /10'8

~l~ U l ~ 17

' !9o 9,,o, g:: (_)

Fig. 5- "Fingerprint" of tryptic peptides from group-specific protein. To the right is a photograph of the tryptic peptides of group-specific antigen after electrophoresis in the horizontal dimension and ascending chromatography. On the left is a tracing of these peptides. Biochim. Biophys. Acla, 154 (I968) 388-396

AVIAN LEUCOSIS GROUP-SPECIFIC ANTIGEN

395

arginine residues of the protein are 22 so that 23 peptides might be predicted on theoretical grounds, assuming that these residues occur in a population of molecules with a uniform primary structure in a unique sequence and assuming classical specicity for trypsin.

Effect of proteolytic enzymes on antigenicity When the effect of proteolytic enzymes on group-specific antigen are compared with their effect on the protein, one concludes that protein integrity is required for antigenicity. One/~g of group-specific antigen was treated with I/~g of trypsin (37 °, p H 7.4, I h) and both trypsin-containing and control tubes were boiled 2o rain to remove trypsin activity. The titer of group-specific activity was 1:64 in the control tube, but undetectable in the trypsin-treated tube. The boiling itself produced a I6-fold fall in titer. When a similar amount of group-specific antigen was treated with o.2/~g of carboxypeptidase (37 °, p H 7.5, I h) and both enzyme and control tubes acidified for i o rain to inactivate the carboxypeptidase, then neutralized, the titer of the control tube was 1:512 and of the carboxypeptidase tube i :256. DISCUSSION

The molecular weight of 23 ooo suggests that group-specific antigen m a y consist of a single polypeptide chain. Conclusive proof that this is true requires a stoichiometric determination of the N- or C-terminal amino acid. In view of the failure here to react with the N-terminus of the protein with FDNB, it is possible that the N-terminal amino group is blocked, as is the case with some other viral proteins. The early release of approximately one equivalent of alanine by carboxypeptidase suggests that this amino acid m a y be the C-terminal group. In addition, the results of tryptic peptide fingerprinting imply a certain uniformity of primary structure, and indicate that there are no repetitive subunits. Assuming this value for the molecular weight, that there are 4 ' lO-1° #g protein per virus particle 27 and that 20% of AMV protein is group-specific antigen ~, one can calculate that there are 2ooo molecules of group-specific antigen protein per virion or per RNA molecule 2s. It will be useful to verify this estimate to help determine the function of group-specific antigen in the virion (e.g., a capsid protein). Group-specific antigen is positively charged even at p H 8.6. Since the number of dicarboxylic residues exceeds the number of dibasic amino acids of the protein, it is likely that an appreciable number of the former is aminated. The high content of proline is of interest because of the effect of this residue on the secondary structure of proteins. No hexosamine occurs in group-specific antigen, although this amino sugar is present in AMV proteins. Evidence from the use of proteases shows that the protein of the group-specific antigen must be intact for the antigen to fix complement with appropriate antisera. Group-specific antigen seems suitable for further study, especially if larger amounts become available and more sensitive means of amino acid analysis can be utilized. Of the various viral antigens, this antigen alone, being group specific, is likely to be practical for protein characterization without preliminary virological efforts toward cloning and typing. This is especially true because of the tendency of avian Biochim. Biophys. Acta, 154 (1968) 388 396

396

I~. W. ALLEN

leucosis viruses to form phenotypic mixtures 2~. It will be interesting to determine if this protein, when isolated from various members of the avian leucosis group, is identical in structure as its antigenic reactivity and immunodiffusion patterns suggest ~0. This would imply that the group-specific antigen represents a com~non gene in this family of oncogenic R N A viruses. ACKNOWLEDGEMENTS

Support was provided by The American Cancer Society, Inc. grant No. E-278E and by U.S. Atomic Energy Contract AT(3o-I)-2643 . The author is indebted to Drs. B. R. BURMESTER and W. OKAZAKIof the Regional Poultry Research Laboratory, East Lansing, Mich., for their assistance in obtaining a large batch of AMV. He is grateful for the advice and assistance of Drs. M. YOUNG AND M. K. RUES in the ultracentrifugation studies, Drs. E. HABER AND F. F. RICHARDS for the amino acid determinations upon the automatic analyser, and to Dr. R. D. MARSHALL for many helpful discussions. Miss LYNI~ R. COZzA, Miss MARY ELLEN MURPHY, and Miss LYNNE TUCKER provided expert technical assistance. REFERENCES

I R. J. I-IUEBNER, D. ARMSTRONG, M. OKUyAN, •'. S. SARMA AND H. C. TURNER, Proc. Natl. Acad. Sci. U.S., 51 (1964) 742. 2 G. I~ELLOFF AND ~). I~. VOGT, Virology, 29 (1966) 377. 3 F. E. I~AYNE, J. J. SOLOMON AND I-1. G. PURCHASE, Proc. Natl. Acad. Sci. U.S., 55 (1966) 341. 4 H. BAUER AND W. SCH~FER, Virology, 29 (I966) 494. 5 H. BAUER AND ~V. SCH~FER, Z. Naturforsch., 2ob (1965) 815. 6 1). W. ALLEN, Biochim. Biophys. Acta, 133 (1967) 18o. 7 J- W. BEARD, 3 d v a n . Cancer Research, 7 (1963) i. 8 E. A. ECKERT, R. ROTT AND \V. SCH~FER, Virology, 24 (1964) 426. 9 D. ~V. ALLEN, Virology, 30 (1966) I. lO O. H. [,O'~VRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. I{ANDALL, J. Biol. Chem., 193 (193I) 265. I t 1). A. YPI-IANTIS, Biochemist~:~, 3 (1964) 297. 12 M. 1i. I{EES AND M. YOUNG, .]. Biol. Chem., 242 (t967) 4449. 13 D. H. SPACKMAN, \¥. [t. STEIN AND S. MOORE, Anal. Chem., 30 (I958) 119o. 14 T. W. GOODWIN AND R. A. MORTON, Biochem..]., 4 ° (1946) 628. 15 D. EXLEY, Biochem. J., 67 (t957) 52. 16 F. SANGER, Biochem. J., 39 (t945) 507 • 17 D. BEALE AND J. I(. WHITEHEAD, in Tritium in the Physical and Biological Sciences, Proceedings of a .~ymposium on Detection and Use, Vienna, Austria, t (1962) 179. 18 M. BRENNER, ,~k, NIEDERWIESER AND G. PATAKI, in A. T. JAMES AND L. J. MORRIS, New Biochemical Separations, D. V a n N o s t r a n d Co., Ltd., L o n d o n (1964) 123. 19 C. B. ANFINSEN, R. I{. REDFIELD, W. L. CHOATE, J. ])AGE AND W. R. CARROLL, J. Biol. Chem., 207 (t954) 2o~. 20 H. STEGIgMANN AND B. LERCH, Analvt. Biochem., 9 (I964) 417 • 21 E. J. COliN AND J. T. EDSALL, Proteins, .4mino Acids, and Peptides as Ions and Dipolar Ions, Reinhold Pub. Corp., New York, 1943. 22 R. L. HILL, Advan. Protein Chem., 20 (1965) 37. 23 E. SCHRAM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (i954) 33. 24 R. H. PURCEI.L, R. A. BONAR, I). ]3LARD AND J. W. BEARD, J. Natl. Cancer Inst., 28 (1962) lOO3 . 25 J.-Y. LIN, C. M. TSUN~ AND H. FRAENKEL-CONRAT, J. Mol. Biol., 24 (1967) i. 26 J. L. BAILEY, Techniques in Protein Chemislry, Elsevier, Alnsterdatn, 1967. 27 R. A. BONAR AND J. \~. BEARD, J. Natl. Cancer Inst., 23 (1959) 183. 28 \~,". S. ROBINSON AND M. A. BALUDA, Proc. Natl. Mcad. Sci. U.S., 54 (1965) 1686. 29 P. G. VOGT, Virology, 32 (1967) 708. 3 ° L. I). BERMAN AND [~. S. SARMA, Nature, 207 (1965) 263.

Biochim. Biophys. ,~#a, 154 (1968) 388 396