996
0. L. SPONSLER, JEAN D. BATH, AND J. W. ELLIS
WATER BOUND TO GELATIN AS SHOWN BY MOLECULAR STRUCTURE STUDIES' 0. L. SPONSLER, JEAN D. BATH, A N D J. W . ELLIS Physical-Biological Laboratories, University of California, Lo8 Angeles, California Received August $1, 1939
While attempting to obtain a dimensional, molecular picture of the living material of organisms, we found ourselves involved in the interrelationships which occur in protein-water mixtures. The water content of protoplasm in an actively growing period of the organism is about 85 per cent, and the protein content about 10 per cent; in a dormant period the water is greatly reduced, often to one-third or even less. This percentage of about 30 to 35 per cent water is also a critical region, experimentally, in the water content of many protein-water mixtures. Investigations of protein hydration have been carried out, to a great exknt, with gelatin-water mixtures which have furnished a wealth of experiment and suggestions concerning these relations (38,39, 40). Confirmation of these is beginning to come out of recent correlative investigations, among which infrared absorption and x-ray studies are playing an important part. When evidence from these physical methods is combined with that from chemical studies, a fairly complete story of gelatin-water relations is obtained. The gelatin molecule is a long polypeptide chain and, as such, may be considered as fairly typical of the primary structure of the simple proteins (41). A small portion of a typical protein chain model built to scale, using known interatomic distances and bond angles (46), is reproduced in figure 1. It has been suggested (38,39, 40) that water will be held only by the constituent groups which contain oxygen and nitrogen atoms. Water relation studies by various methods, such as heat of imbibition (23,44), imbibition pressure (Zl), freezing point determinations and expansion on freezing (4, 31, 43, 45), osmotic pressure and diffusion measurements (35,36), and pressure-concentration curves (39), indicate a restriction in the freedom of motion of the water molecules when lesa than about 35 per cent water is present in the mixture. It seemed reasonable to suppose that the number of water molecules present in this 35 per cent mixture should be approximately equal to the number predicted when the present knowledge of the water molecule (9), of the nature of the oxygen and nitrogen atoms, and of the hydrogen bridge (46) are correlated with recent amino acid analyses of gelatin. 1 Presented at the Sixteenth Colloid Symposium, held at Stanford University, California, July 6-8, 1939.
WATER BOUND TO GELATIN
997
Computations of this nature, but more general in character, have been attempted (39) without, sufficient experimental support concerning the details of water-protein coijrdination. It is now possible to take into consideration reports on investigations which show not only the numerical relationship between the water molecules and the hydrophilic groups, but also the spatial relations and the manner of bonding. I n order to estimate the number of water molecules which may bc coordinated with a gelatin molecule, it is necessary to consider the recent model of the water molecule proposed hy Hernal and Fowler (9) as well as the recent studies on the chemical and physical structure of gslatin. The water molecule may he thought of as a spherical particle in which the oxygeh and hydrogen nuclei lie in a plane passing through the center
F I ~1.. A portion of a protoin chain model showing twelve reaiduos of various sorts attached to the zig-zag backbone. indicated by the horizontal lines. The oxygen atoms have a dot in the center, the hydrogen atoms are small white balls, the carbon atoms %reblack, m d the nitrogen atoms me slightly lighter and have three bonds. of the molecule, as indicated in the diagrams of figure 2, A and R. The oxygen atom has t n o negative residual charges (46) indicated by E and E’ in figure 2, A. These two negative charges and the two hydrogen protons are distrihobed a t the four corners of a tetrahedron; each charge may be active in coordinating another water molecule, as shown in figure 4. X-ray structure analyses of various organic crystitis have shown that this tetrahedral distribution of residual charges is characteristic of the oxygen and also of the nitrogen atom, especially where accompanying hydrogens occiir ( I , 5, 7, 11, 24, 25, 46, 52). These charges may be associated with the formation of a hydrogen bridge (9, 30, 46), as indicated in the diagram in figure 3, whcre the hydrogen proton of one water molecule is shown displac,ed towards the negative residual charge of a second water molecule (91, thus formius the hydrogen bridge. The molecular centers approach to about 2.7 A. A water polymer consist.ing of four water molecules hridged in this manner to a fifth is shown in figure 4.
/ \L' A-
B
A
FIG.2. (A) Diagram of a water molecule showing the plane made by the oxygen and hydrogen nuclei in relation to the two residual negative charges indicated by E and E' (9); (B) diagram showing the dimensional relationship of the oxygen and hydrogen nuclei. i
I
F---2.8A----4
I
p.0"-
I
I.8A- +
FIG.3. Diagram illustrating the formation of a hydrogen bridge between two water molecules (9).
FIG.4. Illustrating the coiirdination of four water molecules to a central molecule through hydrogen bridges, as in ice crystals. This also illustrates the tetrahedral nature of the oxygen atom (9). 998
WATER BOUND TO GELATIN
999
Hydration centers on the protein chain are associated with the various atomic groups in which these two atoms, oxygen and nitrogen, occur (39). Many types of experimental evidence, such as x-ray analyses of crystalline hydrates (25, 47, 51) and studies of solubility curves (29) and of the heat and entropy of hydration of various compounds (lo), have indicated the coordination of water with these groups (30, 37). The number of water molecules that may be coordinated in this manner with the various hydrophilic groups of the protein is given in table 1, where both experimental and theoretical amounts are presented. It has been convenient to consider these hydration centers on the protein chain as separated into two groups,-those on the backbone and those on the ends of the amino acid residues. The total number per molecule obviously depends upon the length of the protein chain; and TABLE 1 Water molecules coordinated with hydrophilic groups HYDROPHILIC O R O W 0
€120
-OH - COOH =O -XHz )NH
*UMBER OF WATER MOLECULE0 (THEORETICAL)
NUMBER OF WATER MOLECULE0 (EXPERIMENTAL)
REFERENCE0 (EXPERIMENTAL)
4
3 5-4
2 3 2 1
since the length for gelatin is known to be variable, an arbitrary choice of 288 residues is used here. In accordance with this, the backbone contains 288 double-bonded oxygens and the same number of nitrogen atoms, in contrast to 94 residues which have polar end groups. The number of potentially available coordination points supplied by the polar groups of the side chains is given in table 2 as 348. Both types of hydration centers on the backbone, Io and )NH, are potentially capable of forming hydrogen bridges to two water molecules, but when space restrictions, resulting from the close approach of atoms in the construction of the chain, are taken into consideration, only one for each group would be a reasonable estimate. Furthermore, in this firm, almost solid gel having about 65 per cent protein and 35 per cent water even this degree of hydration would probably be too great, since a
1000
0. L. SPONSLER, JEAN D. BATH, AND J. W. ELLIS
>
certaiu amount of protein-to-protein cohesion through bridges between )C=O add NH groups (18) is to be expected. An estimate of about 450 to 500 water molecules coordinated to the backbone hm been made. This number is a rather crude estimate which standing alone would have
relatively little value but, when supplemented by x-ray and infrared interpretations, becomes more significant. The total number of coTABLE 2 Composition f gelatin
I
NUYBlB
QRAYB PE1 100 0. D U l QELATIN
AMINO ACID
Glycine. . . . . . . . . . . . . . . . . . . . . . . . . . Alanine . . . . . . . . . . . . . . . . . . . . . . . . .
25.5 8.7 7.1 Aspartic acid. . . . . . . . . . . . . . . . . . . . 3.4 Glutamic acid.,. . . . . . . . . . . . . . . . . 5.8 Serine... . . . ........... 0.4 Proline. . . . . . . . . . . . . . . 19.7 Hydroxyproline. . . . . . . . . . . . . . . . . . 14.4 1.4 Phenylalanine, . . . . . . .... Tyrosine. . . . . . . . . . . . . . . . . . . . . . . . 0.01 Hydroxylysine.. . . . . . . . . . . . . . . . . . 0.3 Cystine . . . . . . . . . . . . . . . . . . . . . . . . . 0.17 Arginine.. . . . . . . . . . . . . . . . . . . . . . . . 8.2 2.94 Histidine.. . . . . . . . . . . . . . . . . . . . . . Lysine. . . . . . . . . . . . . . . . . . . . . . . . . . 5.92 Not accounted f o r t . , . . . . . . . . . . . . 16.14 Total
1
120.08
REFERENCE
NVMBIU
F CO(lUD1-
NUYUEU
)I ETDBA-
OF AMINO ACID
[email protected].*
TION CENTIM ON N D E CHAIN8
NATE WATEU OLmxnzd ON BIDE CBAINM
7 11 1
28 44 3
29
87
12 5 12 17
84 15 36 51
94
348
90
26 14 7 11 1 45 29 2
