DTA and allied physical studies of some synthetic polypeptides

European Polymer Journal, 1970, Vol. 6, pp. 7-16. Pergamon Press. Printed is England

DTA A N D ALLIED PHYSICAL STUDIES OF SOME SYNTHETIC POLYPEPTIDES* D. B.

GREEN,F. HAPPEY and B. M. WATSON

Postgraduate School of Studies in Textile Technology University of Bradford, Yorkshire, England Abstract--It has now become of interest to study the structure of the synthetic polypeptides as they form simple models to use in the study of the more complex protein structure. In a very vide field, this has been done from the point of view of the helical and extended forms of the n and B polypeptide structures, and many polypeptide configurations have been characterized. In this paper, studies are made of the n, r, and oJ forms of the poly-fl-benzyl-L-aspartates and their structural transformations on heating, including the types of energy transforms (endothermic or exothermic) accompanying these modifications. These latter changes have also been followed by X-ray and i.r. studies which have enabled us to characterize the forms unequivocally. In one of the poly-fl-benzyl-L-aspartates, the transformation on heating has been shown to be n ~ ~ ~ r; the n ~ oJ transformation is exothermic and the o, -~/3 endothermic in character. INTRODUCTION THE STUDIES of synthetic polypeptides, particularly those o f the homovariety, ~) have lead to a much clearer understanding o f the structures o f the natural polypeptides. This has been due to the realization that the synthetic structures can readily be used as models in the case of more complex structures of the proteins generally/2) It has been on the basis o f such original work that the helical structures o f a proteins were postulated first by Pauling and Corey ~3) and confirmed later by m a n y workers; ~4-~ X-ray and i.r. techniques were the two major methods by which these studies were carried out. The a and fl forms have been clearly identified and in the main their structures have been generally accepted based in the a case on the PaulingCorey helix and in the latter generally as fully extended molecular pleated sheets. These originally simple structures have also been used as a basis o f the X-ray analysis of the more complex large protein molecular crystals. (s) It has been to some extent difficult for classical protein physicists to understand how the a ~ / 3 transformation in the proteins such as the k.m.e.f, g r o u p could be physically justified in a solid state f o r m / 9 ) In such cases, a five fold spiral has to pull out into an extended molecule with a two fold screw axis. C o n c o m i t a n t with this transformation, the attendant rotation o f the side chain residues must occur to a very marked extent and overcome the steric hindrance f r o m adjacent chains.

a ~ / 3 transformations The conversion o f ~ ~ / 3 forms, both o f which m a y be stable under certain conditions, has been a subject o f physical study by m a n y workers. (~°-x2) In solution the a-helix to r a n d o m coil transformation has been investigated both theoretically and practically and can be induced by p H variations, solvent composition or temperature; the whole o f this field has been reviewed by Urnes and Doty. (~3) A n a ~ / 3 transformation has also been observed on heating alkaline solutions of poly-L-lysine by * Paper presented at the Second Prague Microsymposium on Structure of Organic Solids, 16-19 September 1968. 7

8

D.B. GREEN, F. HAPPEY and B. M. WATSON

Sarkar and Doty. (~4~ In the solid state, transformations from ct ~ ~/structures have been achieved by stretching high tool wt. polypeptides in steam, in some cases the preliminary swelling in formic acid being necessary, tla) In these cases however, the actual thermodynamics of the processes has not been capable of quantitative study and the work threw little or no light on the nature of the transformation. With the advent of the methods of differential thermal analysis and differential scanning calorimetry, however, the quantitative nature and transformation temperatures can clearly be observed in the solid state; tls't#) the molecular configurations can be identified in the polypeptides by X-ray and i.r. measurements from specimens at various temperatures during the transformations. METHODS The polypeptides discussed in this paper are listed in Table 1, along with their sources and average degrees of polymerization. Characterization of the structures of these specimens are defined at various points on the differential thermal analytical graphs by i.r. spectroscopy and X-ray analysis. These are standard techniques and their application in this field has been described previously. ~17~ TABLE l. SAMPI.ES USED IN THIS WORK*

Poly-fl-methyl-L-aspartate(PMLA) Poly-fl-ethyl-L-aspartate(PELA) Poly-fl-benzyl-L-aspartate(PBLA)

Lot No.

DP

AS 19 AS 26 AS 27 AS 20 AS 22 AS 8 AS 10 AS 21 AS 23

100 77 55 77 15 25 45 50

* All samples were obtained from Miles-Yeda Ltd., Rehovoth, Israel. The approximate degrees of polymerization (DP's) are calculated from information provided by the suppliers. The DTA work was carried out using a DuPont 900 Differential Thermal Analyser with the samples either in vacuo or in air. Most specimens were studied at a heating rate of 10°/rain and deviation from this rate, as in Fig. 2, is clearly shown in the diagrams. A DuPont 950 T G A attachment was used for thermogravimetric analysis. The i.r. spectra were obtained using a Grubb-Parsons Model GS2 i.r. spectrometer; the samples were dispersed when in powder form in KBr pressed discs. Powder X-ray photographs were obtained using a fiat plate camera (d = ca. 4 cm) with a Raymax 100 rotating target crystallographic unit using Cu K, radiation. RESULTS Figure 1 shows thermograms of poly-fl-benzyl-L-aspartate (traces 1 and 2), poly-flmethyl-t-aspartate (trace 3) and poly-$-ethyl-L-aspartate (trace 4).

DTA and Allied Physical Studies of some Synthetic Polypeptides

9

Figure 2 shows the thermogram of PBLA (specimen AS10) at varying heating rates; at the slower rate of 2°/min, the shoulder on the exothermic peak at about 100 ° is resolved clearly into a doublet. The complex tracing given by the above specimen (AS10) can be divided into three components A, B and C. A and B are separated by a double exotherm at about I00 ° and B and C by an endotherm at about 180 °. The X-ray diffraction pictures of these samples (taken at A, B and C) are shown in Fig. 3 ; Table 2 gives the measured spacings of these X-ray diagrams. Figure 4 shows the i.r. absorption spectra of PBLA (specimen ASI0), A = unheated specimen, B = after heating to 110 °, C = after heating to 190 °, and Table 3 gives the major absorption frequencies taken from these tracings to establish the molecular configurations of the polypeptides. Figure 5 shows X-ray diffraction pictures of P M L A (specimen AS27), A -----before heating, B = after heating to 170 °, and Table 4 gives the i.r. absorption bands for the above samples.

Exo

t 1

AT

Endo

C4)

60

I

80

I

100

I

120

I

140

Temperature,

I

160

I

180

200

eC

FIG. I. Thermograms (D.T.A.) of polyaspartate esters. (I) PBLA (10 mg sample AS10, AT = 0.2 °) (2) PBLA (3) PMLA (4) PELA (15 mg samples of AS21, AS19 and AS20 respectively. AT = 0" 1°)

10

D . B . GREEN, F. HAPPEY and B. M. WATSON

Exo

Ar

Endo

20

1

~

I

i

T

]

P

r

40

60

80

I(DO

t20

t40

160

180

Temperoture,

200

"C

FIG. 2. Thermograms (D.T.A.) of PBLA (specimen AS10) at heating rates of 2, 5 and 10°/min. TABLE 2. SPACINGS OBSERVED IN X-RAY DIFFRACTION POWDER PHOTOGRAPHS OF SAMPLE AS10, AFTER HEATING TO TEMPERATURES IN THE REGIONS A , B AND C

Region A dob,(,/~) Int. 17 12" 6

4" 7

m (3) vs (a)

mw 3

Region B

do,,,

Int.

17 13" 7 -7" 0 6' 2 5.05 --4.63

m ws

-

vvw vw s

m

-

4.27 4.04 3.79

m m w

--

3" 41

w(br)

3"09

w

2.45 2"30

vw vw

2" 16

vw

d==t=(l~,)

hkl

Region C d,b,(~) Int. 17

13" 80 9"76 6- 90 6" 18 5"05 4" 88 4"75 4" 60 4"36 4.26 4'07 3" 83 3 " 63 3"51 3"45 3"40 3"35 3"13 3" 09 2"91 2" 85 2" 68 2"44 2"30 2- 22 1" 18 2"11

100 110 200 210 101 220 111 300 310 201 211 320 221 301 4O0 311 410 321 420 401 411 421 440 600 441 620 601

4" 7

s

m (3)

dob, are the observed spacings; dc,~= (region B) are based on a tetragonal unit cell with a = b = 13"80.~, c = 5.42/k.

A

B

FIG. 3. X-ray diffraction photographs of PBLA (specimen ASI0}. (A) unheated (B) after heating to 100: (C) after heating to 180 ° (a, o} and/] forms respectively}.

[facin~ p. 10]

FIG. 5. X-ray diffraction photographs of P M L A (specimen AS27).

(A) unheated (B) after heating to 170 '.

ii~!~ ~" ii!!!~i! ¸¸~!?!Ji~!'~i~!!!i~i!!i~!~ ~i~Liiiii~i~

B FIG. 6. X-ray diffraction photographs of PELA (specimen AS22). (A) unheated (B) after heating to 2152,

,I

5.5

8oo

Woveleng'l-h,

6.0

1700

Wove n u m b e r ,

p.

1600

cm -I

6-5

I

500

FIG. 4. Infra-red absorption spectra of P B L A (specimen ASI0) in tile range 1900-1480 cm - ~ . IA) unheated (B) after heating to I00 ° (C) after heating to 180 °.

o

~3

900

~O

I

I00

I

150

t

200

Temperature,

l

250

*C

J

300

L

350

I

.1(10

I

450

°C/min

FIG. 7. Thermograms ( T G A ) o f polyaspartate esters. IA) P M L A (specimen AS27) (B) P E L A (specimen AS22) (C) PBLA (specimen ASI0). Initial sample weight ~ 11 mg in all cases.

c iT,

]

3

O

W

[at"

o

o

>.

I:1 ¢x.

,--I ;>

12

D.B. GREEN, F. HAPPEY and B. M. WATSON

TABLE 3. INFRA-RED ABSORPTION FREQUENCIES (cm -1) OF POLY-fl-BENZYL-L-ASPAKTATE(SAMPLE AS10) IN "mE RANGE2000--667 crn-1

Region A (a-helix)

Region B (o,-helix)

Region C fl-form)

Designation

1738 (vs)

1730 (vs) 1679 (s,sh) 1670 (vs) 1584 (w) 1538 (ms) 1499 (m,sh)

1726 (vs)

Ester carbonyl stretch

1636 (vs) 1582 (w) 1529 (ms) 1498) m,sh)

Amide I Aromatic ¢, (C--C) stretch Amide II Aromatic (C---C) Phenyl At

1663(v~ 1585 (w) 1550(ms) 1499(m,sh) L

J

y-

1294 (m) 1260(ms) 1227 (ms) 1216 (ms) 1167 ~)

1455 (row) 1450 (w,sh) 1415 (w,sh) 1390 (m) 1356 (mw) 1333 (w) 1292 (m) 1265 (ms)

Aromatic (C--C) Phenyl Bt Benzyl CH: scissoring? a--CH, scissoring a--CH~ wagging Benzyl CH2 wagging Tertiary C - - H bending Amide III

1289 (m) 1267 (ms)

1231 (ms) 1216 (ms) 1167 (s)

1215 (ms) 1168 (s)

Ester C--O

1124 (mw, sh) lO81(w)

1081 (w) 1053 (w)

1081 (w) 1053 (w) J

Y

1028 (w) 1002 (m) 965 (m) 908 (mw) 887 (w,sh) 843 (w) 827 (w) 783 (w) L

--

Aromatic Benzene ring } Side-chain absorptions

806 (w) 778 (w)

Y

752 (m) 738 (m) 696 (ms)

"1 Benzene ring C - - H out of .~ plane stretching Aromatic ~ (C--C)

Key: vs = very strong, s = strong, ms = medium-strong, m = medium, mw = medium weak, w = weak, sh = shoulder, sp = sharp.

DTA and Allied Physical Studies of some Synthetic Polypeptides

13

INFRA-REDABSORPTIONFI~QLr~NCIES(cm- ,) OF POLY-~-METHYL-L-ASPARTATEIN THE REGION 2000-700 cm-t. (BANDSCOMMONTO BOTH a AND ~ FORMSARE GIVENIN THE MIDDLECOLUMN.)

TABLE 4.

a form

~ form 1740 (vs)

1664(vs) 1553 (s)

1636 (vs) 1534 (s) 1435 (ms) 1418 (w sh) 1395 (w sh) 1368 (m) 1339 (w)

1304(ms) -1258 (m) 1231 (m) 1220(m) l190(w)

1299 (ms) 1278 (m)

Designation Ester carbonyl stretching Amide I Amide II Methyl C - - H antisymmetric bend a-----CH2 scissoring mode a---CH, wagging mode Methyl C - - H symmetrical bending Tertiary C--H bending Amide III

1217(m) 1171 (s) 1121 (m)

1080 (mw) 1008 (mw)

--

1036 (mw)

(Methyl alcohol impurity) 1020 (m) 998 (s) 911 (m) 899 (s) 874 (w) 850 (m) 806 (w)

784 (w)

Bands due to the side-chain, either to the rocking of the methyl group or the motion of the C----O--C group. 778 (w)

Key: vs = very strong, s = strong, ms = medium-strong, m = medium, mw = medium weak, w = weak, sh = shoulder, sp = sharp.

F i g u r e 6 shows X - r a y diffraction pictures o f P E L A (specimen AS22), A = before heating, B = after heating to 215 °, a n d the i.r. a b s o r p t i o n b a n d s are given in T a b l e 5. F i g u r e 7 shows the t h e r m o g r a v i m e t r i c tracings in v a c u o o f the three aspartic polyp e p t i d e s ; they confirm that the p e a k s in the D T A tracings after a b o u t 220 ° are associated, at least in part, with d e g r a d a t i o n o f the polymers. A t a b o u t 50 °, there is a weight loss associated with the volatilization o f free a n d loosely b o u n d water. This is generally o b s e r v e d in p o l y p e p t i d e s o f this type a n d in proteins, "s~ a l t h o u g h it m a y be a m o r e c o m p l e x tracing if there are traces o f m o r e firmly b o u n d w a t e r which even in v a c u o d o n o t c o m e off until a t e m p e r a t u r e o f 120 ° 150 ° has been reached. The weight loss occurring at a b o u t 150 ° in P M L A (Fig. 7) is due to the loss o f firmly b o u n d methyl a l c o h o l present in the original specimen. The m a j o r peaks however in the specimens, P B L A (AS21), P M L A ( A S I 9 ) , P E L A (AS20), are associated with a t r a n s f o r m a t i o n f r o m a ~ / 3 over a range o f t e m p e r a t u r e s f r o m 120 ° to 160 ° a n d are e n d o t h e r m i c in character. In all these specimens examined, the a ~ / 3 t r a n s f o r m a t i o n was n o t f o u n d to be reversible. On cooling and reheating, the specimens r e t a i n e d their/3 configurations.

14

D.B. GREEN, F. HAPPEY and B. M. WATSON

TABLE 5. INFRA-REDABSORPTIONFREQUENCIES(cm-1) OF POLY-fl-ETHYL-L-ASPARTATEIN TIlE REGION 2000----700 cm-t. (BANDSCOMMONTO BOTH ,, AND fl FORMSARE GIVENIN THE MIDDLECOLUMN) a form

~ form 1740 (vs)

1664(vs) 1553 (s)

1636 (vs) 1530 (s) 1465 (w) 1444(w) 1414 (m) 1399 (m) 1376 (s) 1350 (w) 1339 (w)

1299 (s) -

1300(s) 1280 (m)

-

1255 (ms) 1229 (m) -1212 (m) 1181 (s) 1163 (m) 1124 (w)

Designation Ester carbonyl stretching Amide I Amide II Ethyl mode C.CH3, C - - H antisymmetric bend a--CH2 scissoring a---CH2 wagging C.CH3, C - - H symmetrical bend Ethyl mode Tertiary C - - H bend Amide III

1220 (ms) 1180 (s) 1160(m)

Ester C---O

I044(w)

The rocking of the ethyl side-chain, or the C---O--C group vibtation.

1115 (w) 1096 (m) 1081 (w) 1024 (ms) 975 (w) 890 (mw) 859 (mw) 809 (w) 783 (w)

766 (w)

Key: vs = very strong, s = strong, ms = medium-strong, m = medium, mw = medium weak, w ----weak, sh = shoulder, sp = sharp. DISCUSSION In earlier extensive w o r k on fibrous proteins the k.m.e.f, g r o u p were characterized in one way at least b y their ability to exhibit a reversible ~ ~ / 3 t r a n s f o r m a t i o n u n d e r certain restricted c o n d i t i o n s o f extension a n d contraction/19~ L a t e r such considerations were a p p l i e d in the case o f some o f the synthetic p o l y p e p t i d e s , where u n d e r certain c o n d i t i o n s o f stretching the a p o l y p e p t i d e could be t r a n s f o r m e d to the e x t e n d e d / 3 form. (la'2°'2t) In the case o f the less crystalline c o p o l y m e r s which c o u l d be melted before degrading, the/3 f o r m could be t r a n s f o r m e d into the a form. Such specimens were c o p o l y m e r s o f DL-/3-phenylalanine and DL-leucine a n d o f DL-flphenylalanine, DL-leucine a n d 7-benzyl-L-glutamate.(22) Such/3 ~ ct t r a n s f o r m a t i o n s c o u l d also be o b t a i n e d b y excessive swelling in certain n o n - p o l a r solvents a n d also b y p r e c i p i t a t i o n o f these materials in n o n - p o l a r solvents generally. A t no stage, however, in these extensive experiments was it possible to o b t a i n even qualitatively the energetics o f the t r a n s f o r m a t i o n s . A l o n g with these p r o b l e m s , it was also difficult to envisage an a ~ / 3 t r a n s f o r m a t i o n , p a r t i c u l a r l y reversible in character, involving the extension o f a helical structure with 18 a m i n o acid residues in five turns into a c o m p a r a t i v e l y fully extended chain with a two fold screw axis with the 18 residues in nine turns o f this

DTA and Allied Physical Studies of some Synthetic Polypeptides

15

axis. Further these two chain arrangements had to be capable of reversible transformation. It is, therefore, in this field that the transformations reported earlier are discussed. There are two major components involved in the transformations. (a) The opening of the hydrogen bonds, which in the helix are intrachain in character, to form inter-chain bonds along and generally perpendicular to the chain axis. These generally form in one plane in the form of pleated sheets. (b) The disruption of inter-chain packing of side groups (amino acid residues) uniformly around the helix, and dominating the packing conditions, to inter-chain attachments in a plane perpendicular to that containing the C O - - N H hydrogen bonds. Considering the DTA tracings, it has now been possible to demonstrate clearly that in poly-/3-benzyl-L-aspartate (specimen AS10) the transformation is obtained in "solid state" conditions via an intermediate oJ form. All these forms had been recog~nized previously, (z3) but this appears to be the first indication of such a complex system of transformation. The transformation from a to oJ is exothermic and the thermogram at this stage can be resolved into a doublet at about 100 °. Because of the presence of this doublet, it is reasonable to assume that the transformation may be two stage in character. Here the two stages may b e : - (a) The hexagonal packing changes to tetragonal chain packing. and (b) The a-helix must distort to form a four-fold screw along the fibre axis. It is, however, possible that in this part of the transformation distinct morphological components may play a part e.g. spherulites of different sizes may be present in the sample. It has been suggested that multiple melting peaks observed in nylon may have such an origin. From the DTA tracing (Fig. 2) some evidence of 'pre-melt crystallization' may be observed as a shoulder prior to the co ~ / 3 peak. In the a form the density of the sample was ~ 1.2 g/cm 3 while in the w form the paracrystalline helix crystallizes on a tetragonal lattice and has a density of 1 •3 g/cm 3, calculated from a tetragonal unit cell in which a = b = 13.8 A °, c = 5.42 A". (See Table 2.) After heating to above 180 °, a second transformation occurs from a ~, --->/3 structure. From the DTA tracing, this can be seen to be endothermic. It should be stated, however, that the tool. wt. of the sample is comparatively small for a polypeptide and that the helix is probably in the left-handed sense. (z4) Although this two stage transformation ~--> ~o--->/3 is real, the shorter chain structure may add considerably to the ease of transformation. It is however clear that such a definite route of transformation in a comparatively crystalline polypeptide gives great additional weight to the acceptance of the a --->/3transformation in natural proteins, where the side chain packing is not usually very crystalline. In the case of the three other aspartate esters, benzyl (specimen AS21) ethyl, and methyl, there is in all cases a clear ~---> 13 transformation, endothermic in type; it has not been possible as yet to demonstrate a reversible a ~ / 3 transformation in any of the specimens. When the PBLA (sample AS10) is recrystallized from chloroform, it takes up the configuration. If this sample is then heated, however, it undergoes a weakly endothermic a--+ ~ transformation at 165° without the development of an intermediate o~ structure.

16

D.B. GREEN, F. HAPPEY and B. M. WATSON

In this work, it can be said that the ~---> 3 t r a n s f o r m a t i o n in the synthetic polypeptides considered is an e n d o t h e r m i c reaction. It c a n occur with the f o r m a t i o n o f intermediate crystalline structures (o~) b u t it is clearly obvious t h a t there is sufficient f r e e d o m o f m o v e m e n t to allow the ~ helix to u n w i n d to form a 3 structure. F r o m these p r e l i m i n a r y results it is h o p e d first to e x p a n d the D T A studies to the less crystalline p e p t i d e co-polymers, to establish these various m o l e c u l a r t r a n s f o r m a tions, a n d secondly to study quantitatively the t h e r m o d y n a m i c s o f these t r a n s f o r m a tions in synthetic a n d n a t u r a l polypeptides. Acknowledgements--Two of the authors ('D. B. Green and B. M. Watson) thank the Wool Textile Research Council for financial support during the course of this work. Our thanks are also due to Mr. P. R. Blakey for assistance with the X-ray studies.

REFERENCES (1) (a) C. H. Bamford, W. E. Hanby and F. Hapl~y. Proc. R. Soc. A205, 30 (1951). (b) C. H. Bamford, L. Brown, A. Elliott, W. E. Hanby and I. F. Trotter, Nature, Load. 169, 357 (1952); 171, 1149 (1953). (c) L. Brown and I. F. Trotter, Trans. Faraday Soc. 52, 537 (1956). (2) C. H. Bamford, A. Elliott and W. E. Hanby. In: Synthetic Polypeptides. Chapter 12. Academic Press, New York (1956). (3) L. Pauling and R. B. Corey. Pro¢. natn. Acad. Sci. U.S.A. 37, 241 and 729 (1951). (4) M. F. Perutz, Nature, Lond. 167, 1053 (1951). (5) F. H. C. Crick, Nature, Lond. 170, 882 (1952). (6) W. Cochran, F. H. C. Crick and V. Vand, Acta Crystallogr. 5, 581 (1952). (7) T. Miyazawa and E. R. Bloat, J. Am. chem. Soc. 83, 712 (1961). (8) D. R. Davies, Ann. Rev. Biochem. 36, 321 (1967). (9) F. Happey, Proc. Int. Wool Textile Res. Conf. Australia B153 (1955). (10) K. M. Rudall, Proc. R. Soc. B141, 39 (1953). (11) E. G. Bendit, Nature, Lond, 179, 535 (1957). (12) A. Skertchly and H. J. Woods, Proc. Second Quinquennial Wool Text. Res. Conf. T517 (1960). (13) P. J. Urnes and P. Doty, Adv. protein Chem. 16, 401 (1961). (14) P. K. Sarkar and P. Dory, Proe. hath. Acad. Sci U.S.A. 55, 981 (1966). (15) B. M. Watson, D. B. Green and F. Happey, Nature, Lond. 211, 5056 (1966). (16) B. M. Watson, Ph.D. Thesis, University of Bradford (1968). (1"/) A. Elliott, In: Poly-a-amino acids (edited by G. D. Fasman) Arnold, London (1967); Chapter I and T. Miyazawa, ibid Chapter II. (18) F. Happey and B. M. Watson. Biopolymers 5, 1029 (1967). (19) W. T. Astbury. Trans. Farad. Soc. 34, 378, (1938); J. int. Soc. Leath. Trades Chem. 24, 69 (1940). (20) C. H. Bamford, L. Brown A. Elliott, W. E. Hanby and I. F. Trotter, Proc. R. Soe. B141, 49 (1953). (21) C. H. Bamford, L. Brown, A. Elliott, W. E. Hanby and I. F. Trotter, Nature, Lond. 113, 27 (1954). (22) C.H. Bamford, W. E. Hanby and F. Happey, Proe. R. Soc. A206, 407 (1951). (23) E. M. Bradbury, L. Brown, A. R. Downie, A. Elliott, R. D. B. Fraser, W. E. Hanby and T. R. R. McDonald, J. molec. Biol. 2, 276 (1960). (24) E. M. Bradbury, L. Brown, A. R. Downie, A. Elliott, R. D. B. Fraser and W. E. Hanby, J. molec. BioL 5, 230 (1962).