Aug. 5 , 195s
RAMAN SPECTRA OF CERTAIN AMINOACIDSAND CREATINE [CONTRIBUTION FROM
THE
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BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY ]
Raman Spectra of Amino Acids and Related Compounds. XII. Various Amino Acids Derived from Proteins and Creatine1n2 BY DAVIDG A R F I N K E L 3 RECEIVEDMARCH6, 1958 Raman spectra are reported for serine, threonine, proline, hydroxyproline, valine, leucine, isoleucine, lysine, arginine, creatine and methionine. All were studied in the form of the respective cations, and most in the dipolar ion form as well, but isoelectric serine, leucine, isoleucine and creatine were too insoluble to study in solution as dipolar ions. Proline and hydroxyproline were studied as cation, dipolar ion and anion, and some marked changes, especially in the C-I-I stretching frequencies, were noted when the dipolar ion was converted to the anion. The spectrum of cationic creatine shows some resemblance to that of the methylguanidinium ion, but that of the arginiue cation shows very little. Certain other correlntions between spectra and structure are pointed out.
I n preceding papers of this series we have studied the Raman spectra of ~ y s t e i n ewhich ,~ has an ionizing group (sulfhydryl) of particular significance, and which are SUEof glycine and a- and ciently simple molecules to permit some theoretical analysis. In this paper we shall consider the spectra of a variety of other amino acids which are important because of the part they play in protein structure, but which are too complicated to permit much theoretical analysis a t present. They may be divided into five categories: (1) the hydroxyamino acids, serine and threonine; (2) the imino acids, proline and hydroxyproline; (3) amino acids with non-polar side chains: valine, leucine and isoleucine; (4) basic amino acids: lysine and arginine; (5) the sulfur-containing amino acid, methionine, The Raman spectrum of creatine (methylguanidine acetic acid) will be considered with this group of amino acids, even though i t is not an amino acid, because of its close relation to arginine. The spectrum of histidine has been considered in a previous paper.6 Experimental The techniques of obtaining the Raman spectra have been described .'j The amino acid solutions studied were clarified and freed of fluorescent impurities by treatment with charcoal a t slightly acid pH. Alkaline solutions were prepared from the acid ones using Millipore filters' for clarification. Proline and hydroxyproline slowly decompose on standing, especially when exposed to intense light, to yield yellow fluorescent products. To remove these it was necessary t o boil with charcoal, and a solution allowed to stand several days had to be treated with charcoal again before further study. Fluorescent impurities present in lysine, arginine and creatine were removed by treatment with Amberlite XE-67 anion-exchange resin: the amino acid was dissolved in water, stirred for 45 minutes with resin which had been thoroughly washed with HCl and then with water, and then crystallized by addition of ethanol and ether (this step was omitted for creatine). I t seems likely that picric acid is the impurity removed from lysine, and flavianic acid the impurity removed from arginine, as these are commonly employed in the respective isolation of these compounds. (1) This paper is taken from the P h . D . thesis of David Garfinkel, Graduate School of Arts and Sciences, Harvard University, 1955. (2) Supported in part by a grant (NSF-G621) from the National Science Foundation t o J. T. Edsall. (3) Predoctoral fellow of the National Science Foundation, 19531954. Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. Requests for reprints should be addressed to Dr. J. T. Edsall, T h e Biological Laboratories, Harvard University, Cambridge 38, Mass. (4) D . Garfinkel and J. T. Edsall, THISJOURNAL, 80, 3823 (1958). ( 5 ) M. Takeda, cf al., ibid., 80, 3813 (1958). (6) D. Garfinkel and J. T. Edsall, ibid., 8 0 , 3807 (1958). (7) Millipore Filter Corp., Watertown, Mass.
The nature of the impurity removed from creatine is unknown. Concentrations (reported as weight/volurne, based on the isoelectric compound) of the solutions used for measurement of spectra were: serine hydrochloride, 2970 (isoelectric serine is too insoluble t o study); threonine hydrochloride, 25%; isoelectric threonine, 15%; proline hydrochloride, 36%; isoelectric proline, 45%; proline sodium salt, 37%; hydroxyproline hydrochloride, 23%; isoelectric hydroxyproline, 21 %; and hydroxyproline sodium salt about 15% (using material recovered from the other hydroxyproline solutions, and with K I added as a fluorescence quencher) . The concentrations of the solutions of the larger amino acids, used for measurement of spectra were: valine, 7YG; valine hydrochloride, 22%; leucine hydrochloride, 16%; isoleucine hydrochloride, 19%; lysine monohydrochloride, ZYO; lysine dihydrochloride, 17%; arginine monohydrochloride, 30%; arginine dihydrochloride, 2670; creatine, 14%; methionine hydrochloride, 29%. The isoelectric forms of leucine, isoleucine and methionine were too insoluble to study. All of the above were clarified by filtration through \Vhatman No. 4 paper with charcoal. Methionine sodium salt was prepared by making a 3oY0 solution of the hydrochloride with 2.5y0 KI, filtering with charcoal, as above, adding sufficient NaOH to titrate it to the sodium salt, and then filtering through Whatman No. 4 paper to remove any dust added with the NaOH. The lysine and arginine were obtained from Merck & Co. and from the Mann Laboratories, respectively. All the other compounds were obtained from the California Foundation for Biochemical Research, Los -4ngeles. The amino acids were checked for purity chromatographically.6 All except methionine were free of any ninhydrin-reactive impurity. The leucine preparation was free of isoleucine and v i c e versu. A trace of an impurity which moved like serine was found in the methionine. However, even the strongest serine line is missing from the methionine spectra, and i t is believed that this small trace of impurity has not contributed any false lines to the methionine spectrum.
Results and Discussion Serine.-The Raman spectrum of serine hydrochloride is recorded in Table I. The line a t 1735 can be assigned to a CO stretching motion of the TABLE I" RAMAN SPECTRUM O F SERINE HYDROCHLORIDE, [HOCH*.CH(NHa+) .COOH]Cl680(VW) , 741( VW), 780(2), 811(vw), 831(3b) , 858( w), 907(lb), 974(2b), 1035(Jb), 1093(w), 1134(vw), 1156(vw), 1250(lb), 1305(w), 1359(vw), 1401(vw), 1468(3), 1735(1b), 2870(vw), 2896(1b), 2910(2b), 2969(5b) a Intensities were estimated visually and reported as 1,2,3 ... for increasing intensities, except that the faintest are reported as weak (w) or very weak (vw). Broad lines are designated b, very broad as vb. No correction has been made, in this or the subsequent tables, for concentration of the amino acid solution from which the spectrum was obtained. (8) T h e assistance of Dr. R . H. McMenamy is gratefully acknowledged.
DAVIDGARFINKEI,
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carboxyl group, and that a t 1035 is likely to be due to a C-O-H deformation frequency of the alcoholic hydroxyl (based on the findingg of such a deformation frequency in alcohols a t 1020 cm.-l). The line a t 1468 ern.-' can be assigned to a C H deformation motion. The 2896 frequency may be due to stretching of the tertiary C-H bond on the a-carbon atom, those a t 2910 and 2969 to the methylene stretching motions of the -CH20H group. Threonine.-The Raman spectra of threonine are listed in Table 11. It is noteworthy that the line a t 1047 cm.-', assigned to an OH deformation motion, appears in the infrared spectra of threonine, lo in aqueous solution (as well as in the solid) but not in the infrared spectrum of threonine in DzO. This is to be expected if the given assignment is correct, since the hydrogen on the alcoholic hydroxyl exchanges readily with deuterium. The spectra of both serine and threonine show only one C H deformation line, in contrast to the larger amino acids which have two. TABLE I1 RAMMAN SPECTRUM OF THREONINE HOCH(CH8)CH(NH3+)coo Isoelectric
Hydrochloride
492(vw)
497(vwb) 559(vw b) 623(vwb) 748(2b)
Assignments
851(wb) 868( wb) 894(vwb) 934(1b) 998(vwb) 1047(wb)
1106( wb) 1252(vw) 1349(2b) 14n5(3b) 14
