322
M. ASAI,M. TSUBOI, T. SHIMANOUCHI AND S. MIZUSHIMA
VOl. 59
INFRARED SPECTRA OF POLYPEPTIDES AND RELATED COMPOUNDS. I BY MASATOMO ASAI,MASAMICHI TSUBOI, TAKEHIKO SHIMANOUCHI AND SAN-ICHIRO MIZUSHIMA Contribution f r o m the Chemical Laboratory, Faculty of Science, Tokyo University, Hongo, Tokyo, J a p a n Received August B Y , 1964
Infrared absorption spectra of nine polypeptides have been measured in the frequency region from 5000 to 600 cm.-'. There have been observed many bands common to all these polypeptides, but some of the bands appearing between 1200 and 800 cm.-I were found to depend considerably on the sequence of amino acids in the polypeptide. It was concluded that in silk fibroin glycine and alanine residues are arranged alternately, although some authors prefer the at-random arrangement to the periodic arrangement.
Many papers have been published on the infrared spectra of polypeptides and proteins.lJ There are, however, many problems remaining to be solved as to the interpretation of the spectra in terms of the structure of polypeptides. Concerning these problems we have published several papers on the structure of molecules with one and two peptide bonds, (N-methylacetamide,a acetylamino acid, Nmethylamides,4 etc.). As Professor Noguchi of Kanazawa University kindly placed various samples of synthetic polypeptides at our disposal, we could extend our structural studies and could obtain some interesting results concerning the sequence of amino acid residues in polypeptides. The results will be reported in this and the following communications. Experimental Noguchi found a new method of synthesis6 of polypeptides by the olymerization of carbothio henylamino acids or carbothiopienyloligo peptides. The l r s t seven samples shown in Table I were prepared by him by this method. I n addition to these the last two samples shown in the same table were used in the present measurement. SAMPLES USEDI N
TABLE I INFRARED MEASUREMENT
THE
Sample
Mol. wt.
Polyglycine 135,200 Poly-m-alanine 22,400 11,500 Copolymer of glycine and DL-alanine (1 :1) Polygly cyl-DL-alanine 34,800 Poly-nbphenylalanine 59,400 Poly-8-alanine 43,500 Poly-glycyl-nL-phenylalanine 30,600 Silk fibroin (crystalline part), see ref. 17 .... Poly-e-capramide film .... The infrared spectra were recorded at 20" in the frequency region from 5000 to 600 cm. -l by the Baird spectrophotometer with NaCl optics. Except for poly-e-capramide film the samples were measured in Nujol mull.
Results and Discussions The results of infrared measurements are shown in Figs. 1-4. The dotted lines indicate the absorption of Nujol bands overlapping the bands of the sample under investigation. Figure 1 contains the absorption curves of N-methylacetamide in the liquid state, and acetylglycine N-methylamide, (1) G. B. B. M. Sutherland, Advances in Protein Chem., 7 , 291 (1952). (2) L. J. Bellamy, "The Infrared Spectra of Complex Molecules," Methuen, London, 1954, Chapter 12. (3) S. Mizushima, T. Shimanouchi, 8. Nagakura, K. Xuratani, M. Tsuboi, H. Baba and 0. Fujioka, J . Am. Chem. Soe., 7.2, 3490 (1950). (4) S. Mizushims, T. Shimanouchi, M. Tsuboi, et aE., ibid., 79, 1330 (1951); 74, 270, 4639 (1952); 76, 1863 (1953). (5) J. Noguchi, J. Chem. Soe. Japan, 74, 961 (1953); J. Noguchi and T. Hayakawa, J . Am. Chem. Soc., 76, 2846 (1954).
acetylglycylglycine N-methylamidea and polyglycine in powder form. At the top of the figure is given the assignment of the bands of N-methylacetamide to various vibrational modes. Figure 2 shows the absorption curves of polyDL-alanine and poly-DL-phenylalanine, Fig. 3 those of poly-&alanine and poly-e-capramide and Fig. 4 those of glycine-DL-alanine copolymer (1:l), polygly cyl-DL-alanine (periodic), crystalline part of silk fibroin and polyglycyl-m-phenylalanine (periodic). It will be seen from these figures that there are several bands common to all polypeptides measured in this experiment. These appear at 3300, 3080, 1640, 1550, 1480-1340, 1280-1230 and 725 cm.-l. On the other hand the bands observed in the range from 1200 to 800 cm.-l are characteristic of the structure of each polypeptide and, as shown below, we can derive some interesting conclusions from the frequency values of their peaks. I. Absorption Bands Common to All Polypeptides Measured in This Experiment. (a) The Band at 3300 cm.-'.-This arises from the NH stretching vibration of the peptide bond in the planar trans form which is involved in the hydroI
gen bonds, - h H .
I
. . O=C-N-H
,
. . O=C-,
in the chain type polymer, The details have deen rep~rted.~J On the higher frequency side of this band there was sometimes observed a shoulder peak due to the adsorbed water which naturally disappears on dehydration. For example, poly-e-capramide under 0.01 atmosphere of water vapor showed the water band a t 3500 cm.-l which disappeared on dehydration over concentrated sulfuric acid.8 On the other hand the sample of poly-0-alanine used in this experiment had bound water which could not be removed on dehydration over concentrated sulfuric acid.9 For the disappearance of the water band it was necessary to heat the sample for ten hours at 135" in the dry air over phosphorus pent oxide. (b) The Band at 3080 'cm.-l.-This is also assigned to the bonded NH stretching v i b r a t i ~ n . ~ , ' ~ The intensity ratio of this band to that at 3300 cm.-' depends on the nature of the polypeptide. (c) The Bands at 3000-2800 Cm.-l.-They arise from the CH stretching vibrations. (6) The sample was prepared by T. Sugita in our Laboratory. (7) M. Tsuboi, Bull. Chem. SOC.Japan, 2.2, 215, 255 (1949). (8) M. Tsuboi, i b i d . , 26, 160 (1952). (9) The measurement was made with Perkin-Elmer Infrared Spectrophotometer Model 112, with LiF prism. (10) S. Mizushima, T. Shimanouchi and M. Tsuboi, Nature, 166, 406 (1950); M. Tsuboi, Bull. Chem. Soe. Japan, 25, 385 (1952).
SPECTRA OF POLYPEPTIDES AND RELATED COMPOUNDS
April, 1955
323
.
Fig. 3.-Infrared
WkYE 1ENtTH X,U
WAVE L t i i m iu r absorption curves of poly-p-alanine (A) and poly-e-capramide (B).
.
.'
Fig. 1.-Infrared absorption curves of N-methylacetamide (A), acetylglycine N-methylamide (B), acetylglycylglycine N-methylamide (C) and polyglycine (D). Dotted lines indicate the absorptions due to Nujol in which the samples are suspended ( v , stretching; 6, deformation; P, out of plane vibrations).
.
I
,
,
;
"
I
,
,
,
'
,
"
,
,
,
,
'
, n
W%if
lf/m /N A .
Fig. 4.-Infrared absorption curves of glycine-nL-alanine at random copolymer (1:1) A; poly-glycyl-Dbalanine (periodic) B; crystalline fibroin C; and poly-glycylnL-phenylalanine D. WAYf
LFN6JH /N
/u
.
Fig. 2.-Infrared absorption curves of poly-nL-alanine ( A ) and poly-DL-phenylalanine (B).
is assigned (d) The Band at 1640 Cm.-'.-This to the C=O stretching vibration. is charac(e) The Band at 1550 Cm.-l.-This teristic of the peptide -NH-CO- bond which is involved in N-H . . . O=C hydrogen bonding and in which the N-H and C=O bonds are in the trans position with respect t o each other. This band has been assigned either to the N-H deformation vi-
brationll or to the C-N stretching vibration.12 It has been shown by Fraser and Price13 and by Miyazawa, et aE.,14 that the two vibrations contribute considerably to this band. are (f) The Bands at 1480-1340 Cm.-'.-These assigned to the various C-H deformation vibrations. (11) R. E. Richards and H. W. Thompson, J . Chem. Soc., 1248 (1947). (12) H. M. Randall, R . G. Fowler, N. Fuson and J. R. Dangl, "Infrared Determination of Organic Structure," D. Van Nostrand Co., New York, N. Y. (la) R . D . B. Fraser and W. C. Price, Nature, 170, 490 (1952). (14) T. Miyazawa, e l at., to be published.
324
M. ASAI, M. TSUBOI, T. SHIMANOUCHI AND S. MIZUSHIMA
(g) The Twin Bands at 1280 and 1230 Cm.-i.Most of the polypeptides show these bands. From the comparison of the four absorption curves in Fig. 1 the twin bands can be concluded to correspond to the 1290 cm.-' band of N-methylacetamide, assigned to the vibration contributed by both the N H in-plane deformation and C-N stretching motions.'4 It is not probable to assign these bands to the NH3+ vibration of the side chain. In Fig. 5 are reproduced the absorption curves in this frequency region. It will be seen that the intensity ratio of these two bands is different in different polypeptides.
Vol. 59
capes detection for those in which no two glycine residues adjoin each other. (3) This band is observed for glycine-alanine copolymers in which the two residues are arranged at random and which, therefore, contain glycylglycine structures. This conclusion is compatible with the experimental results of Blout and Linsley who observed a strong band at 1015 i 10 cm.-l for glycylglycine, oligopeptides with glycine residues only and polyglycine. l6 In Table I1 are shown the bands of various substances observed in the frequency region, 1015 f 20 cm.-'. It will be seen that acetylglycine Nmethylamide and acetylglycylglycine N-methylamide also show a sharp and strong band a t 1030 cm.-' similar to the 1015 band referred to above (see also Fig. 1, B,C). Such a band does not appear at all for acetylnorleucine N-methylamide and acetylvaline N-methylamide. 111. Sequence of Aminoacid Residues in Polypeptides.-The appearance or otherwise of the band a t 1015 cm.-l referred to above is a good criterion upon which we can test the existence of two consecutive glycine residues in the polypeptide. I n addition t o this there have been observed some other bands which seem to depend upon the sequence of amino acid residues. The lines shown in Fig. 6 denote the positions and intensities of bands observed in the region from 1200 to800cm.-l. It is seen that the spectrum of glycine-alanine a t random copolymer (1 :1) are almost the superposition of those of polyglycine and polyalanine, including the band a t 1015 cm.-' of polyglycine and that a t 965 cm.-' of polyalanine. The latter may be characteristic of the alanylalanine structure just as the former is characteristic of
(I)
GIL
I
LE) An
.
130QcM.-/ /ZJQ
1200-1300
cm.-' region absorptions of polypeptides.
( W)(-t-4-In
II
I
I I
I
.I
m
III
I
L IIH (6 Aln
Fig. 5.-The
n
I,
I/
(h) The Band at 725 Cm.-'.-This arises from N-H out of plane deformation ~ i b r a t i 0 n . l ~ 11. The Band at 1015 Cm.-' Characteristic of the Glycylglycine Residue.-As referred to above the spectra observed in the region from 1200 to 800 cm.-l are characteristic of the structure of polypeptides and, therefore, the interpretation of the absorption bands in this region is very important. Among others that of the band a t 1015 cm.-' is clear-cut. This band is concluded to arise from the glycylglycine structure from the following reasons : (1) This band is very strong and sharp for polyglycine and is not observed a t all for such polymers as poly-@-alanine and poly-m-alanine which do not contain glycine residues. (2) Even in polymers containing glycine residues this band es-
By contrast in glycine-alanine periodic polymer in which the two different residues are arranged alternately along the polymer chain, the two bands a t 1015 and 965 cm.-l are not observed a t all and the two strong bands appear at 998 and 975 cm.-'. There has been observed no other significant difference between the spectra of the at-random copolymer and periodic polymer. In connection with this, the spectrum of '(the crys-
(15) H. K . Kessler and G. B. B. M. Sutlierland, J . Chem. P h y s . , 21, 570 (1953).
( 1952).
(P)S/fX
I
I,
,
II
, I ,I
,
Fig. 6.-The 8 0 0 ~ 1 3 0 0cm.-l region absorptions of polyglycine (G,,), poly-DL-alanine ( An), glycine-DL-alanine at random copolymer ( I :1) ( G,A)n), poly-glycyl-m-alanine (periodic) ((G-A),,), and silk fibroin.''
(10) E. R. Blout and S. C. Linsley, J . Am. Chem. Soc., 1 4 , 1946
April, 1955
LIGHTSCATTERING
THEBANDSIN
TABLE I1 REGION 1015 i 20 C M . - ~
THE
Compds.
Formula
N-Methylacetamide
325
BY A Q U E O U S SOLUTIONS OF SODIUM L A U R Y L S U L F A T E
CH3CONHCH3
Carbothio phenylglycine
C~H~SCONHCH~COOH
Glycylglycine Triglycine N Hexaglycine Acet ylglycine-N-methylamide Acetylglycylglycine N-methylamide Polyglycine
NHa +CHnCONHCH&OO+?JHaCHzCO(NHCH&0)1,4NHCHzCOOCH3CONHCHzCONHCHa CHaCONHCHzCONHCH2CONHCHa (-NHCHnCO-), CHa
Intensity
Cm.-1
.....
None /lo12 \ 1003 1015 1015 1029 1030 1015
Medium Weak Strong16 Strong16 Strong Strong Strong
Carbothiophen yl-DL-alanine
CeH5SCONHAHCOOH
1023
Medium
Poly-Dbalanine Carbothiophenyl-p-alanine Poly-p-alanine
(-NH HCO-)n CeHhSCONHCHnCHzCOOH
None 1023 None
Weak
r
Carbo thiophenyl-glycyl-m-alanine
(l90;:
Poly-glycyl-DL-alanine (periodic) Glycine m-alanine copolymer (1 :1) (at random)
(-NHCH2CO-)(-NH
talline residue of silk fibroin”“ is interesting. This shows strong bands a t 996 and 976 cm.-l but no bands a t 1015 and 965 cm.-l. We can, therefore, conclude that in the crystalline part of silk fibroin the glycine and alanine residues are arranged alternately just as in periodic polymer and not as in at(17) 8. Akabori, K. Satake and K. Narita (Proc. Japan Acad., 2 6 , 206 (1949)) showed that silk fibroin underwent gradual degradation in dilute hydrogen peroxide at rooni temperatures, in the presence of a small amount of ferrous sulfate as oatalyaer, leaving insoluble residue, 35% in weight of the original silk fibroin, which was found to be a high polymer consisting mainly of glycine and alanine (1 : 1). This residue was called b y them the “crystalline residue” and was considered by them to correspond to Meyer’s crystalline part of silk fibroin because i t showed sharp Debye-Scherrer rings similar to those shown h y the original silk fibroin. The sample used in the present experiment is this crystalline residue of silk fibroin.
..... .....
Weak Weak
998
Medium
1015
Medium
random copolymer. This is in agreement with the model of silk fibroin proposed by Meyer, et aZ.,ls but not with that corresponding to the at-random copolymers of glycine and alanine. Acknowledgment.-The authors wish to thank Professor J. Noguchi of Kanazawa University for the samples of many synthetic polypeptides used in this experiment. They are also indebted to Professor S. Akabori of Osaka University and Professor IS.Narita of Ochanomizu University for the sample of crystalline residue of silk fibroin and t o Dr. K. Hoshino and Dr. H. Yumoto of Toyo Rayon Company for poly-c-capramide film. (18) K. H. Meyer, M. Fuld and C. Klemm, Helv. Chim. Acta, 28, 1441 (1940).
LIGHT SCATTERING BY AQUEOUS SOLUTIONS OF SODIUM LAURYL SULFATE’ BY JOHNN. PHIL LIPS^ AND KAROL J. MYSELS Department of Chemistry, University of Southern California, Los Angeles 7 , California Received December 6 , 1064
M9surement.s of turbidity of ure sodium dodecyl sulfate in water and in salt solutions are reported.
Their interpreta-
tim 1s briefly discussed and lea$ to several conclusions: The molecular weight of the micellea varies from 23,000 to over
%,oOO aa the concentration of salt increases. The effective charge of a micelle, calculated For the first time from light ‘*catbring, remains constant a t about 14. The turbidity in water at high detergent concentration can be predicted on the basis of very simple assumptions suggesting that the micellar charge and size may not change radically in this region. Traces of lauryl alcohol induce the formation of micelles below the C.M.C. A t still lower concentrations they giye rise to large turbidities which may be used to detect the presence of lauryl alcohol and also to determine its solubility. Finally, some of the previous light scattering results are reviewed and reinterpreted.
Light scattering measurements of solutions of association colloids are a very powerful tool for (1) Presented in part a t the J. W. McBain Memorial Symposium of the Division of Colloid Chemistry a t the Chioago Meeting of the American Chemical Society, September, 1953. (2) Colgate-Palmolive Peet Company Postdoatoral Fellow, 19621953.
probing their nature. AS has been shown by Deb ~ e , ~they , 4 provide information about the weight of micelles and as We shall show, also about micellar charge, and about the presence of Certain impUritieP. (3) P. Debye, Ann. N. Y . Aced. 8ci., 6 1 , 573 (1949). (4) P, Debye, Tms JOURNAL, 68, 1 (1949).
