1126
A. M. BUBWELL, K . F. KAEBB AND W. H. RODEBUBH
(33) RODEBUSH, W. H.: Chem. Rev. 19, 59 (1936). (34) RUSRELL, J. L., AND RIDEAL,E. K. : Proc. Roy. soc. (London) MW,540 (1936). (35) SCHALEK, E., AND SZEGVARI, A.: Kolloid-Z. Sa, 318 (1923);33, 326 (1923). (36) SCEMIDT, C. L. A. (Editor): The Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois (1938). (37) SPONSLER, 0.L., AND DORE,W. H.: Ann. Rev. Biochem. 6, 63 (1936). (38) SVEDBERG, T.:Chem. Rev. 10, 81 (1937). (39) SVEDBERG, T.: Ind. Eng. Chem., Anal. Ed. 10, 113 (1938). (40) WERNER,H.:Ber. 62B, 1525 (1929). (41) WRINCH, D.M.:R’ature 137, 411 (1936);198, 241 (1936);139, 972 (1937). (42) WRINCH, D.M.: Phil. hlag. 26, 313 (1938). (43) Wn, H.: Chinese J. Physiol. 5, 321 (1931).
THE INFRARED ABSORPTION OF PROTEINS IN THE 3 fi REGION. XII‘ A. M. BUSWELL, KARL F. KREBS, AND W. H. RODEBUSH Department of Chemistry, University of Illinois, Urbana, Illinois Received July 3, 1940 INTRODUCTION
.
The study of bound water in biological tissues has been handicapped from its inception by the weaknesses inherent in the methods used for the determination of bound water. None of the various methods gives any clue as to the mechanism of bonding, and all are subject to criticism on the basis of the fundamental assumptions involved (12). A possible mechanism for the bonding of water was suggested by Buswell in 1928 (3). His suggestion that water mas bonded to gelatin by means of the hydrogen bond followed from a correlation of the paper by Latimer and Rodebush (17), who suggested the hydrogen-bond hypothesis, and the classic work in spectroscopy of Coblentz (9), who had noticed peculiarities in the infrared absorption of water a t 1.5 p when present as “water of constitution” in certain minerals and in a gelatin film. The work of Wulf and Liddel (21, 13), who showed that the first harmonic of the hydroxyl group disappeared when opportunities for hydrogen bonding were present, indicated that the infrared absorption technique offered a method of testing this proposed mechanism, and also gave promise of being adaptable to quantitative determination of bound water in biological hydrogels. 1 Presented at the Seventeenth Colloid Symposium, held at Ann Arbor, Michigan, June 6-8, 1940.
INFRARED ABSORPTlON OF PROTEINS
1127
In a previous publication (6) a curve was given for the absorption spectrum for gelatin in various stages of hydration. Absorption bands were found at 3.37, 3.2, and 3 p. At that time the 3.4 was assigned to the HC vibkation, and the 3 p band, since it varied widely with water content, was assumed to be a measure of bound water. Although the
5Y
FIQ.1. Absorption curveB for gelatin. Curve 1, air-dried; curve 2, dried-at 102OC. for 2 hr.; curve 3, dried at 152'C. for 4 hr. 3.2 band appeared to vary somewhat with the water content, it was tentatively assigned to 0-H-N or N-H--N bonding. Recent work now rules out the possibility of NH-N (10). On the replotting of our data (see figure l), placing the height of the 3.37 bands a t the same elevation in all cases (a procedure which we now find to be justified), there appears to be practically no variation in the
1128
A. M. BUSWELL, K. F. KREBS AND W. H. RODEBUBH
3.2 band with water content and no change in the height of the 3 p band between specimens dried a t 102’ and 152OC. Curves of specimens of appreciable moisture content show a distinct broadening or shoulder a t the shorter wave length, a t about 2.90 p . I t also appears that the suggestion of Ellis and Bath (11) that “the absorption at 3 p . . . in a gelatin specimen dried for 14 hours a t 122OC. is caused by protein S H groups rather than by residual water” is a valuable one and a t least partially correct. It must be pointed out, however, that the SH frequency itself is relatively weak, whilc the 3 p band in dry gelatin is very strong. It is further known that the HN frequency is much shorter than 3 p , namely, 2.83 to 2.93 (7). However, as in the case of water, the shift to longer frequency with bonding results in greatly increased intensity of absorption. This 3 p band now appears to be due to a linear KHO bonding plus the effect of absorbed water. Thc hydration of proteins, as shoxn in this earlier work as well as in that on zinc insulinate as presented in this paper (figure 2), is evidenced by greatlN increased absorption in the 3 k region, with distinct broadening and in some cases a shoulder a t approximately 2.90 p . Owing to the complexity of the NH and OH absorption spectra in the bonded state, it has been necessary to determine the various bonding frequencies of these groups in simple organic molecules and also to work out their absorption spectra when present in such complicated structures as the proteins. This work is reported elsewhere (10). The present work is then a report of the infrared absorption of a series which includes memben of several of the classes of proteins based upon solubilities. EXPERIMENTAL
The spectrometer used was one of the prism type, built in this laboratory in 1936. The source of radiation was a street-lamp bulb, which was supplied with a current of 7 amperes from the 110 D.C. line balanced against six 2-volt storage batteries to avoid line fluctuations. A more detailed discussion of the instrument is available in a previous publication (8). The materials studied were all too insoluble in suita,ble solvents to permit study in the solution form, and so a method for obtaining them in the form of a thin film was devised. The solution of the protein was evaporated on a 24 s 50 mm. microscope cover slide floating on mercury. The use of 0.5 cc. of a 1 per cent solution of the protein in water, 0.6 per cent ammonium hydroxide, or 70 per cent alcohol, depending upon the properties of the protein, gave a film of the desired thickness. Films built up by the Langmuir technique (2, 16) were also tried, but were abandoned in favor of the simpler deposition method when no appreciable qualitative differences in the two types of film were found. The
1129
INFRARED ABSORPTION OF PROTEINS
*methodwould have a distinct advantage in quantitative determinations, in that the molecular absorption coefficient could be calculated from the known number of layers. The preparation of such films would be laborious, since they would have to be about five thousand layers in thicknas. In the hydration studieb on two of the proteins, gelatin and insulin, a
-
21 N C I NSULINATE
1749
I
I
I
I
FIQ. 2. Absorption curves for zinc insulinate. Curve 1, over barium chloride solution; curve 2, over magnesium nitrate solution; curve 3, air-dried; curve 4, over phoaphorue pentoxide.
cell provided with air ducts to permit the passage of air arross the film was used. This arrangement made it possible to regulate the degree of hydration of the film without removing it from the absorption cell. Different degrees of hydration were effected by passing the air through saturated salt solutions of known vapor pressure.
1130
A. M. BUBWELL, K. F. KXEBS AND
w. E. RODEBUSH
Data on the molecular extinction coefficient of absorbed water are necessary to complete the solution of the problem. It has been impossible to obtain such data with the experimental technique outlined above, for the following reasons: the material is very opaque and the absorption due to water is very intense. It is therefore necessary to use very thin films in order to permit the passage of sufficient energy to obtain satisfactory galvanometer readings. The actual change in weight of a 24 x 50 mm. film between different degrees of hydration is very close to the TABLE 1 Absorption bands i n sizteen proteins ABBOBPTION PEASE (Wnvm LWQTE IN p )
coypoIRpD
Gelatin. . . . . . . . .
2.91
Zein. . , . , , . . . . . . . . . . . . . . , , , , . . . . Gliadin, , . . . . . . . . . . . . . . . . , . . . . . . Hemoglobin Insulin. . . . . . . . . . . . . Hordein. . . . . . . . . . . . . . . . . . . . . . . . . Pepsin. . . . . . , . . . . . . . . . . . . . . . . . , . Salmine, . , . , . . . . . . . . . . . . Soybean protein. . . . . . . . . . . . . . . . . Ficin. . , , , . , . . . . . . . . . . . . . , . . . . . . Casein. , , . . . . , . . . . . . . . . . . . . . . . . , Egg albumin Glucose, , . . , . . . . . . . . . , . . . . . . . . . . Agar. . . , , , . . . . . . . . . . . . , . . . . . . . . . , ., , . . .. . , . . . . . . . . . . . . .B-Amvlose Glycyl-dl-alanine. . . . . . . . . . . . . . . . . a-Bromoisocaproylleucine. . . . . . . . Alanine.. . , , , , . , . , . . . . . . . . . . . . . . , Proline. , , . , . . . . . . . , . . . . . . . . . . . . . Urea, . , , . , . . . , , . , , . . . . . . . . . . . . . . N-Butylurea. , . . . . . , . , . . . . . . . . . . . Thiourea.,. . , , . . . . . . . . . . . . . . , . . . N-Phenyl-", N'4ibutylthiourea. ,
2.85 2.79
3.00 2.97 2.97 2.99 2.99 2.99 3.00 3.00 3.01 3.01 3.01 3.01 3.02 3.02 3.02 3.03 2.96 2.92 2.96 3.03
2.97 2.99 snd 3.04 2.97 and 3.04 2.96 3.03 3.05
3.33 3.34 3.38 3.35 3.34 3.35 3.35 3.35 3.35 3.35 3.36 3.34 3.35 3.34 3.34 3.34 3.40 3.35 3.39 3.09 3.22 3.36 3.23 3.35 3.23 3.40 3.45 3.34 3.14 3.22 3.22 3.20 3.21 3.21 3.22 3.22 3.21 3.22 3.09 3.20 3.22 3.20 3.20 3.22 3.22 3.22
2.91
3.36 3.42 3.12 3.24
3.36 3.43
limit of error of even a microbalance. A large number of attempts to determine molecular extinction coefficients of absorbed water, a t both 1.44 and 3 p , have been unsuccessful. RESULTS
The absorption bands found in the sixteen proteins studied are given in table 1, together with the data obtained in the study of certain other related compounds.
INFRARED ABSORPTION OF PROTEINS
1131
DISCUSSION
A . Proteins The interpretation of the absorption peaks found in the proteins is based upon considerable experimental data obtained from the study of simple organic nitrogen compounds, as mentioned above (10). The most logical interpretation of the results obtained from the study of compounds having a linkage of the amide type -C-TS-R
/I
I
O H is that the absorption found at 3.00 p is due to a single "-0 bond bonds in and that the band a t 3.22 p arises as a result of double "-0 a ring-dimer structure -C--X-R
i l
O H
H O I I
R-N-C-
Gelatin and peptone (figure 3, curves 1 and 2) show relatively strong absorption a t 3.22 p , the band being distinct from the broad 3 p band. This band resembles the absorption a t 3.22 of N-ethylacetamide (7). The natural proteins show much less pronounced absorption a t 3.22. This is shown by a low peak in glutenin, ficin, zein, and gliadin (figure 4, curves 3 to 6) or by a mere shoulder on the 3 p peak in the case of egg albumin, rennet, edestin, and soybean protein (figure 5 , curves 7 to 10). In the case of acid casein and rennet casein (figure 6, curve 11) there is more pronounced absorption a t 3.22 in the former than in the latter. Also, among the natural proteins there is a distinct indication that the less drastic the method of isolation the less pronounced is the absorption a t 3.22 p. This cumulative evidence suggests a relation between the structure related to this band and the phenomenon of denaturation. As mentioned above, this 3.22 band is associated with the ring-dimer structure. In nature one might expect that in the presence of excess water it would bond to half the hydrophilic groups, thus R-C-N-C
/I 1
0 H*OHt H O.HOH
I
C-3-C-R
II
1132
A. M. BUSWELL, K. F. KREBS AND W. H. RODEBUSH
preventing ring formation. This water may be considered as “water of constitution.” At the same time the natural products contain a large and variable amount of water which may be removed by mild desiccation and may be reabsorbed or reimbibed repeatedly. But on severe dehydra‘tion the water of constitution would be irreversibly removed and the
3. GLUTENIN 4. FICIN
5. ZEIN 6. GLIADIN
FIG.3. Absorption curves for gelatin and peptone FIG.4. Absorption curvea for glutenin, ficin, rein, and gliadin
ring structure, which i s very stable, would be formed. The resulting material would be less soluble and leas active in many ways. The above is not put forward as a complete theory of denaturation but only as a suggestion of a mechanism of part of the process. The appearance of the 3.22 and 3.00 I( bands in all of the proteins studied
INFRARED ABBORPTION OF PROTEINS
1133
suggests the important r61e that the hydrogen bond plays in the structure of the proteins. These data afford experimental justification to the postulates of protein structures involving hydrogen bonds which have been made by several authors (18, 14, 15). The spiralled beta-keratin structure suggested by Seurath (19) appeals to the authors as being highly probable, in that it would appear to permit
11-1.
CASEIN
- ACID -
11-2. C A S E I N R E N N E T 12. SALMINE
u FIG.5 FIG.6 FIG.5. Absorption curves for egg albumin, rennet, edestin, and soybean protein FIG.6. -4bsorption curves for acid casein, rennet casein, and salmine a closer approach of adjacent polypeptide chains and thus allow hydrogen bonding of the ring-dimer type between the peptide links. This type of linkage would explain the appearance of both the 3.22 and 3.00 p bands. The spiralled structure might be compared to two twisted threads with the peptide links as the points of contact. Of course, for fully extended polypeptide chains the situation is different.
1134
A. M. BUSWELL, K. F. KREBS AND
w.
H. RODEBUSH
Salmine is a member of the protamine class of proteins; these yield on hydrolysis comparatively few amino acids, among which the basic ones predominate. Salmine, obtained from salmon sperm, contains 87 per which has, cent of arginine, NHzC(=NH)NHCHZCH&HZCHNHZCOOH, in addition to the usual amino group, three extra nitrogen-hydrogen groups, one of which is an imino group. The typical protein, on the other hand, is made up of many different amino acids. Only a very small per cent of these amino acids contain NH groups other than the amino group. One would expect, therefore, that salmine would show an absorption spectrum different from that obtained from most proteins. The band a t 3.09 p appearing in the absorption spectrum for salmine (figure 4, curve 12) is unique in the protein spectra so far obtained. It undoubtedly arises as a result of the large excess of nitrogen-hydrogen groups in the protein. One might predict that XH-0 bonds involving the imino groups would appear a t a wave length differing from the ordinary Y H - 0 absorption. These KH--O bonds could be formed either through the -NH, or the =NH groups of the amino acid side chains. Since this wave length is not far from the absorption at 3.16 p in glycine and a t 3.14 p in unsubstituted acetamide, it seems probable that it is to be assigned to association through -NH2 rather than =”. The need for further study of this protein and of simpler compounds containing the imino linkage is indicated. I t is quite probable that very interesting structures could be postulated for salmine, if more exact data as t o the type of bonding were available. One is naturally concerned with the possibility of nonpeptide-nitrogen bonds in other proteins. I n going through the structures of the amino acids which have been found in the proteins. one finds five amino acids containing non-peptide nitrogens : Tryptophan, (J--JCHC , HNH~COOH
Histidine,
HC==CCH&HNH,COOH
I
HN
1
K
‘CG Lysine, NHsCH~CH~CHzCH~CHNHzCOOH Arginine, NHzC=NHNHCHZCH~CH~CHNHZCOOH Citrulline, NHzCONHCHZCH~CH~CHNH~COOH Of these amino acids tryptophan is rarely present in amounts greater than 3 per cent, histidine is also low in most proteins, lysine varies from 0
INFRARED hBSORPTION OF PROTEINS
1135
to 8 per cent in the proteins considered here, the presence of citrulline in most proteins has not been verified, and arginine varies from 1 to 15 per cent in the usual protein. The characteristics of the spectrum of salmine should be apparent in the spectrum of thymus histone, which contains 27 per cent of arginine, 9.7 per cent of lysine, and 5.8 per cent of histidine (20). I t can be seen that the hydrogen bondings due to the presence of these amino acids in the ordinary protein would be only a very small contribution to the total bonding due t o the peptide linkage. Since the amino acids are probably arranged in some sort of periodic arrangement in the proteins (I), it would not be logical to assume that very many of these extra NH groups would be close enough together in the peptide chain to permit bonding. The absorption of the free N-H groups in the protein would probably resemble that of the primary and secondary aliphatic amines. This type of X-H absorption has been shown to be very weak in intensity (4), and therefore the presence of these few free amino groups, e.g., lysine, might very well be missed in the spectra of the proteins. The effect of the small amounts of histidine and tryptophan occurring in certain proteins cannot be detected in the absorption spectra with the present methods. One would expect, therefore, that there would be a great similarity in the absorption spectra in this region. Peptone (figure 3) also shows a peculiarity in the infrared absorption, in that it has a distinct shoulder on the short-wave side of the 3.00 p band. This peak, if separated from the 3.00 p band, would occur a little below 2.9 p. It appears likely that the band represents unbonded NH groups (10) in the protein fragments that make up the peptone. This might be expected in such a mixture of components, since there would be lack of orientation and complete randomness of arrangement. So far in this discussion no mention has been made of differences appearing in the CH absorption of the proteins due t o differences in the sidechain residues. The reason for this is that the CH band is not very prominent, and no significant observations can be made either as to prominence or exact location of the absorption maxima. The only differences detectable would be in the relative amounts of aliphatic and aromatic carbon-hydrogen, but the relative percentages of those amino acids having aromatic rings is fairly constant in the proteins and the percentage is also relatively low. Bonding between hydroxyl or carboxyl groups in proteins is possible, but resolving power available does not permit their observation. Their number would be small in comparison t o bonds involving nitrogen. Structural linkages other than the hydrogen bond between chains are not detectable in the 3 p region. Of these proposed linkages the disulfide might be exiected to appear a t a wave length greater than 10 p, and the
1136
A. M. BUSWELL,
K. F. KREBS AND W. H. RODEBUSH
ester or salt linkages between acidic and basic amino acids might be detected in the carbonyl region at 5.8 p.
B. Hydration studies The bonded frequency for water-like hydroxyl groups has been shown to appear at a wave length slightly lower than 3 p (5). The results from the study of the carbohydrates support these data, in that the bonded frequency occurs at 2.96 p. The superposition of the absorption due to structural hydrogen bonding and of the absorption of bonded water hydroxyls make hydration studies on the carbohydrates impracticable in the 3 p region. In theqroteins these two types of bonding are separated to some extent, since the predominant type of structural hydrogen bonding involves the NH groups which show absorption in the bonded state at slightly longer wave length than the hydroxyl groups do. The shoulder that appears on the short-wave side of the 3 p band (figures 1 and 2) gives a qualitative picture of water bonding, but the separation is not great enough for any quantitative measurements with the present technique and instrument. It is hoped that improvements in the weighing technique and the use of an instrument of greater resolving power will enable a quantitative determination to be made in the future. SUMMARY
It has been shown that: 1. The proteins are characterized by absorption a t about 3.4 p (HC), at 3.22 p (believed to be due to a ring formed by two peptide links bonded NH to 0), and at 3 p (a broad band due to linear NH to 0). 2. Salmine shows additional absoiption a t 3 . 0 9 ~(attributed to excess "2). 3. The band at 3.22 I( is much weaker in "unaltered" natural proteins than in gelatin, etc. It is suggested that this explains in part the mechanism of denaturation. 4. Hydration causes a broadening of the 3 p band with a shoulder at about 2.9 p. REFERENCES (1) BERGMANN, M . : Harvey Lectures 31, 37 (1936). (2) BLODQETT, K. B.: J. Am. Chem. SOC.67, 1007 (1935). (3)BUBWELL, A. M.: The Chemistry of Water and Sewage Treatment, American Chemical Society Monograph No. 38, pp. 53, 80. Reinhold Publishing
Corporation, New York (1928). (4) BUSWBLL, A. M.: Unpublished data from this laboratory. (53 BUSWELL, A. M., DEITZ,V., AND RODEBUBH, W. H.: J. Chem. Phys. 6, 501
(1937). (6) BUSWELL, A. M., KREBS,K. F., AND RODEBUSH, W. ET.: J. Am. Chem. soc. 69, 2603 (1937).
ULTRACENTRIFUGATION STUDIES ON VIRUSES
1137
(7) BUSWELL, A. M.,RODEBUSH, W. H., A N D ROY,M. F.: J. Am. Chem. SOC.80, 2444 (1938). (8) CLAUESEN, W. F. : Thesis, University of Illinois, 1939. (9) COBLENTZ, K.K.:J. Franklin Inst. 172, 309 (1911). (10) DOWNING, J. R.: Thesis, University of Illinois, 1940. (11) ELLIS,J. W.,AND BATH,J. D.: J. Chem. Phys. 6,108 (1938). (12) FISHER, H.: Thesis, University of Illinois, 1936. (13) HILBERT,G.E., WULF,0. R., HENDRICKS, S. B., AND LIDDEL,U.: J. Am. Chem. SOC.68,548 (1936). (14) HUGQINS, M. L.: J. Am. Chem. SOC.81, 755 (1939). (15) HUGGINS,M. L.:Paper presented at Ninety-ninth Meeting of the American Chemical Society, held a t Cincinnati, Ohio, April, 1940. (16) LANQMUIR,I.: Science 8S, 76 (1937). (17) LATIMER, W.M., AND RODEBUSH, W. H.: J. Am. Chem. SOC.41, 1419 (1920). (18) MIRSKY,A. E.,AND PAULING, L.: Proc. Natl. Acad. Sci. U. 5. 22,439 (1936). (19) NEURATH, H.: J. Phys. Chem. 44,296 (1940). (20) SCHMIDT, C. L. A. (Editor): Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois (1938). (21) WULF,0.R., AND LIDDEL,U.: J. .4m.Chem. SOC.67, 1464 (1935).
ULTRACENTRIFUGATIOK STUDIES OK TOBACCO MOSAIC AND BUSHY STUNT VIRUSES' MAX A. LAUFFER Department of Animal and Plant Pathology, The Rockefeller Instztute for Medical Research, Princeton, New Jersey Received July 3, 1940
The plant viruses which have thus far been obtained in a highly purified state may be divided into two groups,-those which are rod-shaped and those which are essentially spherical. Of the first group, tobacco mosaic virus (20) is the easiest to study, for it is highly stable and may be isolated as a crystalline protein in comparatively large quantities. Of the second group, only tomato bushy stunt virus (2) is available in crystalline form. Much information is available concerning the physical properties of both of these materials. Tobacco mosaic virus solutions exhibit strong double refraction of flow (ll), electrical double refraction (IO), and anomalous viscosity ( i ,19). Concentrated solutions give rise to microtactoids which separate out into liquid crystalline phases (3). The material crystallizes in the form of rod-like para-crystals of microscopic size (20), and the virus particle itself is a rod-like body from 12 to 15 mp l Presented a t the Seventeenth Colloid Symposium, held at Ann Arbor, Michigan, June 6-8, 1940.
