24
ALBRECHT, SCHNAKENBERG, DUNN AND MCCULLOUGH SUMMARY
A polarimetric study of the equilibria between formaldehyde and l(+)-glutamic acid is reported. The values 38.0 and 43.7 were found for the association constants of glutamic acid with 1 and with 2 moles of formaldehyde, respectively. REFERENCES (1) FRIEDEN, E. H., DUNN,M. S.,
CORYELL, C. D.: J. Phys. Chem. 46, 215 (1942). (2) FRIEDEN, E. H., DUNN,M. S., AND CORYELL, C. D.: J. Phys. Chem. 47, 10 (1943). (3) LEVY,M.: J. Biol. Chem. 99, 767 (1933). AND
QUANTITATIVE INVESTIGATIONS OF AMINO ACIDS A S D PEPTIDES. XI1 STRUCTURAL CHARACTERISTICS OF SOMEAMINOACIDS' GUSTAV ALBRECHTI, GEORGE W. SCHNAKENBERGS, MAX S. DUSN, AND JAMES D. RfcCULLOUGH
Department of Chemzstry, Universzty of Californza, Los Angeles, Calzfornza Received September 21, 1948
Knowledge of the structure of amino acids is of the greatest importance in attacking the problem of protein structure. Complete structures have been determined, however, only in the case of glycine (I), &alanine ( l l ) , and the closely related substance, diketopiperaeine (4). Because the difficulties of x-ray crystal analysis increase rapidly with the number of atoms in the molecule, it seems desirable to conduct preliminary investigations of the structural characteristics of other amino acids. A number of amino acids were surveyed in this manner by Bernal (2) in 1931. Five amino acids, not investigated previously, are treated in the present paper. Photomicrographs are shown for three of these amino acids and for four others the structures of which have been investigated by other authors. 1 The authors are indebted to Professors Linus Pauling and 0. L. Sponsler, in whose laboratories part of the x-ray measurements were made. Some of the data in this papei are taken from the thesis of G. Albrecht, which was presented to the Graduate School of the University of California in partial fulfilment of the requirements for the degree of Doctor of Philosophy, June, 1941. This work was aided by grants from the University of California and the Rockefeller Foundation. For the preceding paper in this series see Frieden, Dunn, and Coryell: J. Phys. Chem. 47, 20 (1943). 2 Present address: Department of Chemistry, Pomona College, Claremont, California. 3 Present address: Van Camp Sea Food Company, Terminal Island, California.
INVESTIGATIONS O F AMINO ACIDS A N D PEPTIDES.
XI1
25
PREPARATION O F AMINO ACID CRYSTALS
The crystals were grown by cooling warm saturated solutions of amino acids and by evaporating saturated solutions at room temperature. In most cases single well-developed crystals were obtained only after much persistence. The crystals examined goniometrically were 0.1 mm. to 1 cm. in diameter, but only those of the smaller size were used for the x-ray work. PHOTOMICROURAPHIC TECHNIQUE
The photographic problem was somewhat difficult because of the diameter, thickness, number, and arrangements of facets, and the lack of photographically uscful color and opacity contrast of the crystals. For these reasons macro, rather than the less difficult micro or gross, photography was required. A low-power binocular wide-field Spencer No. 56 microscope was found to meet the magnification requirements. A macroscopic magnification range of 9 x , 15.3X, and 20.7X was obtained by 9X oculars used in conjunction with 1x, 1.7X, and 2.3X paired objectives. The photographic equipment, which was built around the microscope, consisted essentially of a rigid base and a vertical optical bench on which the camera carriage, the base plate for the microscope, and the incident illuminating device could be raised and lowered. A 2t x 3 t in. film-pack camera with shutter but without lens was used. A sliding back was constructed so that the viewing ground glass and film holder could be interchanged. The position of the film plane above the microscope was determined by the field size permitted by the film area. Since thc film was covered by the field at a distance less than 10 in. above the microscope, the photographic magnifications (6.5X, lO.OX, and 15.3X) were somewhat less than the microscopic magnifications. Two methods of illumination were used in conjunction with each other. The transmitted light source was a 200-watt tungsten lamp. The light was first diffused through a ground-glass screen and then passed through a simple Abbe condenser fitted with a dark-field stop. Quarter sectors of Wratten gelatin filters, A (red) and B (green), were placed 90' apart between the two lens cells of the condenser. It was possible to arrange the crystal so that each individual face was illuminated by either red, green, or white light, all against a dark background. Eastman commetcial panchromatic film was used. In order that the gray tones of the photograph would exhibit the same relative brilliance of the crystal faces, a Wratten K3 filter was used to give exaggerated orthochromatic correction. When this illumination was arranged satisfactorily as viewed in the ground glass, it was extinguished and incident light provided by means of four 32-candlepower bulbs placed around and at about 34 in. from the crystal. The average exposure for all magnifications was about 3 sec. The incident light was employed during only about one-third of the exposure time. Even though the lowest magnification possible was used in order to have the greatest depth of focus, it was never possible to have all of the planes in focus
26
ALBRECHT, SCHNAKENBERG, DUNK AND MCCULLOUGH
a t one time. I n order that the crystal might be manipulated in the field of illumination, it was mounted on a large cover slip with a trace of beeswax. The cover slip was held by a clamp and was movable in all directions. Since the tubes of the binocular microscope were a t the normal convergence angle of So, the camera carriage was made adjustable so that the film plane could be made to coincide with the microscopic image. This made it possible to photograph separately the right and left images which, when printed as positive transparencies, could be observed in three dimensions in a stereoscopic viewing box. METHODS USED IN STUDYING AMINO ACID CRYSTALS
After the crystal had been examined for symmetry, it was mounted on an optical goniometer and readings were taken for all faces. From these observations symmetry properties (point group), axial ratios, and interaxial angles were usually determinable. The cleavage properties of the crystals were studied by noting the mode of fracture. The latter is important, as it gives a clue to the orientation of the molecules within the crystal. In the x-ray studies Laue, rotational, and oscillation photographs of single crystals were prepared and from these data the symmetry properties of the crystal (space group) and the size of the smallest repeating unit (unit cell) were determined. EXPERIMENTAL OBSERVATIONS
dl-Valine: It was shown by goniometric examination that dl-valine is monoclinic hemimorphic hemihedral with axial ratios a : b : c = 0.235: 1 :0.244, with p = 70'58'. Cleavage was excellent parallel to 010. Laue photographs showed twenty-four first-order reflections with no systematic absences in hkl, indicating a primitive lattice. Oscillation photographs showed OkO to be absent when k is odd. This observation indicates that there is a twofold screw axis parallel to b and that the crystal belongs in space group Ci - P21. The dimensions of the unit cell, calculated from layer-line dat: and high-order pinacoid reflections, are uo = 5.20 bo = 22.12 co = 5.41 A. (p = 70" 58'). The density of dl-valine, 1.316 g. per cubic centimeter, given by Cohn et al. (3), was confirmed by the flotation method. These values lead to four molecules in the unit cell (calculated, 4.00). dl-Threonine: The flat six-sided plates of dl-threonine were shown by goniometric examination to be orthorhombic hemimorphic hemihedral. The pyroelectric effect with liquid air was not pronounced, but negative results in this experiment are not conclusive. The lack of a symmetry center was deduced from the observation that there are but four molecules per unit cell and the fact that point group D2hwould require a minimum of eight for asymmetric molecules such as threonine. The axial ratios obtained are a : b : c = 1.675:1:0.6644. Rotation Photographs showed layer lines which lead to the unit cell dimensions a. = 13.64 A, bo = 7.75 cO = 5.16 A. The density was found to be 1.437 g. per cubic centimeter. These data correspond to four molecules per unit cell (calculated, 3.99). Laue photographs taken along the three axes showed this
w.,
B.,
w.,
FIG.1. a-Glycine: (a) X 9 ; (b) X 15.3. a-Glycine (5, 12) crystallized readily from water It is monoclinic holohedral, point group C 2 h , with pronounced basal cleavage (parallel t o 010). X-ray studies have revealed thFt it belongs to space group C:, - P21/n. The dimensions of the unit cell are ao = 5.10 A., bo = 11.96 A,, co 5.45 A,, withp = 111'38'. The density is 1.607 g. per cubic centimeter (3) and there are four molecules per unit cell (1, 3,
-
9, 10).
FIG.2. dl-Alanine (X 20.7): dl-Alanine is crystallized with difficulty from water (2, 6). It is orthorhombic hemimorphic hemihedral, point group CI,. The space group is Ciu P b n and there are four molecules per unit cell. The density is 1.40 (11). The dimensions of the unit cell are ao = 12.04 A., bo = 6.04 A., co = 5.81 A. (11). FIG. 3. &valine (X 15.3) FIQ.4. &Serine (X 20.7) FIG.5. dZ-Threonine (X 5) 27
FIG.6. dl-Aspartic acid: (a) X 9 ; (b) X 20.7. Groth ( 7 ) described two forms of inactive aspartic acid, “r-asparaginsaurem rhombisch bisphenoidisch” and ”inaktiv (racemisch) asparaginsaure, monoklin prismatisch.” The crystals obtained in the present work by crystallizing dl-aspartic acid from water were all monoclinic holohedral (monoclinic prisniatic by Groth’s nomenclature) and checked the constants of Groth’s “innktiv asparaginsaure.” The axial ratios determined from the present authors’ goniometric measurementswere a : b : c = 2.098:1:1.245, a i t h p = 86”3’. FIG.7. l(+)-Glutamic acid (X 9): This amino acid, crystallized from water, forms flat four-sided plates and also polyhedra with many faces. The crystals were all orthorhombic enantiomorphic hemihedral. The axial ratios found were a : b : c = 0.6894:1:0.8551. These values compare favorably v i t h a : h : c : = 0.6868:1:0.8548, given by Groth (8); and those reported by Bernal (2) for the enantiomorph, d(-)-glutamic acid. Bernal also gave the space group V4212121 [D: - P2121211, and four molecules per unit cell of dimensions a. = 7.06 H., bo = 10.3 H., co = 8.76 A. 28
INVESTIQATIONS OF AMINO ACIDS AND PEPTIDES.
XI1
29
to correspond to a possible unit of structure. There were no systematic absences of the type hkl in over two hundred firsborder Laue reflections, thus indicating a primitive lattice. No systematic absences were found to occur in hkO, h01, or Okl to space group C,: Pmm. dl-Serine: Goniometric examination showed dl-serine to be monoclinic holohedral with axial ratios a : b : c = 1.194:1:0.522, with fl = 73'47'. Groth (5) reported axial ratios which can be reconciled with those listed above by a different choice of the a- and c-axes. The present authors' choice of axes is based on the habit of their crystals, although either description is correct. Layer-line measurements and calculations from high-order pinacoid reflections show that the unit cell has the dimensions a+ = 10.74 b., bo = 9.15 b., co = 4.78 b. (p = 73'47'). The density was found to be 1.537 g. per cubic centimeter. These data lead to four molecules per unit cell (calculated, 4.00). Absences of h01 with h odd and OkO with k odd indicate a glide plane, a, and a twofold screw axis alongb, giving the space group C L P21/a. (If the axes of Groth had been employed the space group would have been Ci,, - P21/a, since the glide would then have been along a diagonal instead of along the a-axis.) dbNorleucine: It was found by goniometric examination that dl-norleucine is monoclinic holohedral with axial ratios a:b:c = 3.46:1:2.10, with fl = 75'17'. The unit cell dimensions obtained from rotation photographs were a0 = 16.60 b., bo = 4.62 b.,co = 10.0 b. (p = 75'17'). The density was found to be 1.169 g. per cubic centimeter. These data lead to four molecules per unit cell (calculated, 4.04). The absence of OkO with k odd and h02 with 1 odd indicates a twofold screw axis and a glide plane c. These data place dhorleucine in space group C t - P21/c. dLMethionine: This amino acid is very difficult to crystallize, but usable crystals were obtained. It was shown by goniometric examination that dlmethionine is monoclinic hemimorphic hemihedral with axial ratios a :b :c = 2.09: 1 :3.43, with fl = 102'7'. Very pronounced cleavage exists parallel to 001. Rotation and oscillation photographs lead to the unit cell a+ = 9.92 b.,60 = 4.73 A., q = 16.20 b. (p = 102'7'). The density was found to be 1.340 g. per cubic centimeter. These data correspond to four molecules per unit cell (cal-, culated, 4.03). The presence of 010 uniquely determines the space group, which is C+ - P2.
-
-
SUMMARY
Photomicrographs, densities, axial ratios, space groups, unit cells, and number of molecules per unit cell are given for nine amino acids. Five of the amino acids have been investigated for the first time by the present authors. REFERENCES (1) ALBRECHT, G.,AND COREY, R. B.: J. Am. Chem. 800. 61, 1087 (1939). (2) BERNAL,J. D.:Z. Krist. 78, 363 (1931). (3) Corn,E. J., MCMEEKIN, T. L., EDEALL, J. T., AND WEARE,J. H.: J. Am. Chem. 600. 66, 2270 (1934). (4) Corny, R. B.: J. Am. Chem. 600.80. 1698 (1938).
30
J. P. HOLLIHAK AKD D. R. BRIGGS
(5). GROTH,P. : Chemische Krystallographie, Vol. 3, p. 98. ’Iv. Engelmann, Leipzig (1910). (6) Reference 5, page 124. (7) Reference 5, page 276. (8) (9) (10) (11) (12)
Reference 5, page 407. J.,A N D LENEL,F. V . : Z. Krist. 77, 427 (1931). HENGSTENBERG, KITAIGORODSKII, A. I.: J. Phys. Chem. (C.R.S.S.) 8,756 (1936). LEVY,H . A,, AXD COREY,R . B.: J. Am. Chem: Soc. 63,2095 (1941). SCHABCS: Referred to in Jahresber. (Liebig-Kopp) 1864, 676.
VISCOMETRIC ESTIMATION OF PARTICLE DIMENSIOKS. I1 MICELLAR
CHANGES
INVOLVED I N THE HEAT-BODYING O F TUNGOIL‘
J. P. H O L L I H A Y
AND
D. R. BRIGGS
Diuision of Agricultural Biochemistry, University of X n n e s o t a , St. Paul, Minnesota Received Xo$ember 6 , 1942
The earliest workers proposed that the thickening which results when a drying oil is bodied is due to a general colloidal association or to a general polymerization. In either case all of the glyceride molecules mere supposed to be involved. I t is nom held that the reaction responsible for the thickening process does not take place uniformly throughout the material, so that even after gelation a substantial portion remains in the liquid state (4, 5 , 13, 19). This liquid is presumably entrapped and immobilized Trithin a solid network, which is envisioned by the “colloidal-association’’school to be a sort of sponge held t’ogether irregularly by secondary valence forces and by the “polymer” school to be a regular structure held together by primary valence forces (1, 2, 3, 11, 20). It is possible that these two concepts may tend to lose their identity when applied to the gelation process proper, since it is generally agreed that association becomes more pronounced as three-dimensional polymerization progresses. The literature contains considerable evidence to the effect that the bodying process takes place in successive stages (6, 18, 19). According to this supposition, the primary stages result only in a small and regular increase in viscosity, whereas the gelation process proper is due to a rapid secondary reaction, which takes place at the gelation point. The extent of chemical reaction occurring during this hypothetical second stage is still highly controversial (18). iUolecular-.iveight changes during the bodying process have been studied viscometrically by Rolff (21), Kurz (12), and Elod and Mach (8). In the present viscometric study somewhat more detailed knowledge was sought, in 1 Paper S o . 2040, Scientific Journal Series, Minnesota Agricultural Experiment Station. This paper is condensed from a thesis presented by J. P. Hollihan to the Faculty of the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of Doctor of Philosophy, June, 1940. 2 Present address: American Viscose Corporation, Marcus Hook, Pennsylvania.
