18 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS been attempted. The present paper discusses some of the problems that still prevent full clarification of the role of crosslinks in determining keratin structure, and reports on progress of a method for mathematically isolating various components from the amino acid analyses of whole keratin fibers. THE LOCATION OF CROSSLINKS AND SWELLING The two-phase structure for keratins introduced by Feughelman (4,5) solidified for some time the assumption that nearly all aqueous swelling occurs in low-modulus matrix proteins and very little in the high-modulus microfibrils. An alternative model by Menefee (6,7) proposed a reversal of the modulus roles of the matrix and microfibrils, and also supported the idea of an ordered globular matrix, later substantiated by Spei (8,9) however, the assumption of matrix swelling was retained. Studies by Heidemann and Halboth (10), extended by Spei and Zahn (11), have strongly suggested that most swelling takes place in the microfibrils (presumably on hydrophilic side chains). Since crosslinks are so intimately involved in the valuable physical behavior of fibrous kera- tins, the extent of their involvement in a swelling phase is a crucial question.. Unfor- tunately, in spite of efforts of many years, the proper definition of a fully adequate model is still elusive. Experiments by Spei and Zahn (11,12) on the dry and wet intermicrofibrillar distances in three keratins illustrate the ambiguity of the keratin model. As seen in Table I, there is an inverse correlation between the amount of matrix and the increase in wet swelling in going from low to high sulfur keratins. Spei and Zahn interpret this by postulating that the swelling occurs chiefly in the micro fibrils. This is a reasonable idea, since the average residue side chain in the low-sulfur fraction is much more hydrophilic than in the matrix with its high proportion of cystine. On the other hand, Fraser et al. (13) claim that their method for estimating microfibril diameters in similar swollen keratins shows a microfibril wet swelling for porcupine quill of only 11%, with a matrix swelling of 53%. To account for the data of Spei and Zahn, Fraser et al. (16) present a complex and probably unnecessary mechanism. At least a portion of the differences in microfibrillar swelling spacing with different keratins is due to the effect of variations in matrix crosslink density. We can estimate the cystine content for mohair and human hair resulting from increasing the proportion of matrix from that in porcupine quills. On one extreme, if we assume that all the cystine is in the matrix (calculated to be 22.4% half-cystine for the quill), and that the increase in the total area is proportional to the squared ratios of the dry spacings, we can estimate what the cystine levels should be in mohair and human hair. As seen in the last column of Table I, they are too low even for this favorable calculation, indicating need for either a higher crosslink density in these fibers or for another higher cystine component. Choice between these alternatives is more uncertain if there is significant variation in the proportion of intramolecular-versus-intermolecular crosslinking. In a previous study (14) based on a theory of the effect of crosslinking on partial solubilization by hydrolysis, a wool sample was estimated to have 40% of its crosslinks in intramolecular form. Although other keratins were not investigated, variation in the amount of intramolec- ular-versus-intermolecular crosslinking would not be surprising. Additional indirect

COMPONENT DISTRIBUTIONS IN KERATINS FROM AMINO ACIDS 19 Table I Changes in X-ray Spacing of Swollen Keratins (11, 12) Intermicrofibrillar 1/2 Cystine + Spacing, nm Cysteine, % Volume % Sample Matrix Dry Wet Obs Calc Porcupine quills 37 7.8 9.3 8.3 (8.3) Mohair fibers 42 8.2 9.6 10.3 9.6 Human hair 54 9.2 9.8 15.3 12.3 evidence (15,16) indicates considerable intramolecular crosslinking in fibrous keratins. The proportion of intermolecular corsslinks will have the greatest effect if swelling occurs in the matrix, since microfibrils have few disulfide crosslinks of either kind. Careful examination of spacing changes of fibers with different inter/intramolecular crosslink ratios, or of partially reduced and stabilized fibers, may offer an experimental approach toward clarifying the location of aqueous swelling. Taking into account the above ambiguities, we may attempt a sketch of a plausible model for crosslinking in fibrous keratins. Aqueous swelling may occur about as much in the crystalline slightly-crosslinked microfibril side chains as in the amorphous highly- crosslinked matrix. After cleavage of crosslinks, the matrix becomes more hydrophilic owing to the presence of sulfhydryl or cysteic acid groups. Within the matrix, crosslinks are about equally divided between intramolecular and intermolecular. Although cross- links between adjacent cystine residues are unlikely (17), the next favored configuration (16, 18) is of the form-Cys-X-Pro-Y-Cys-, with an intramolecular crosslink con- necting the two cystines. An earlier interpretation (19) of the amino acid analyses of the insoluble gel fraction remaining after partial hydrolysis of wool fibers (20) suggests that threonine, serine, valine, arginine, and glutamic acid will most often occur in the X and Y positions. This is borne out by sequence studies by Swart (21). Although intramolecular crosslinks do not directly contribute to mechanical properties, they may act as a reservoir for renewing intermolecular linkages. This is particularly important when considering how a reduced fiber may be set into a new configuration after reox- idation of sulfhydryl groups. THE MAJOR KERATIN COMPONENTS METHOD How many independent component groups are needed to define the composition and behavior of a keratin fiber? As mentioned previously, a fibrous keratin contains upwards of 100 distinct proteins, each belonging, at least partly, to some conformation family-- alpha helical chains, randomly amorphous regions, or globular structures. These pro- teins are held together by disulfide crosslinks, hydrogen bonds, and other lesser links. Attempts to build a model from this myriad detail are likely to be unsuccessful. Association of the high-sulfur fraction with the matrix, and the low-sulfur fraction with microfibrils, is currently well established. The high-sulfur (H) fraction exhibits the greatest diversity of protein content. The low-sulfur (L) fraction is much less complex. The cuticle (C) also contains a range of proteins, but on the whole resembles