360 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS below it in the fiber bundle. In this short length of fiber there is no opportu- nity for the preferentially stained half of the fibers to "switch sides." Accordingly, if the next section showed preferential staining of the opposite half of the corresponding fibers, when dyed with an acid dye, the views of Ohara, Horio and Kondo, and Mercer would be confirmed. Dusenbury and Coe (2) have performed experiments of this sort, and they have found, in- stead, that the acid dye preferentially stains the same side of the fibers as the basic dye. Photomicrographs of such dyed successive sections are shown in Fig. 4. These sections were prepared with a Hardy-type microtome, using Neg- O-Lac as the embedding medium. One section (Fig. 4a) was dyed with Janus Green B, a basic dye, thirty seconds at 75øC., and rinsed with distilled water. The section prepared from the next successive cut of the the microtome (Fig. 4b) was dyed with an acid dye, Formyl Violet S4B, thirty seconds at 75øC., and rinsed with distilled water. The same result has also been observed when wool fibers were embedded in a block of plasticized polymethylmethacrylate and successive cross sections were prepared with a Spencer sliding microtome and then dyed, alternately, with basic and acid dyes. This demonstrates that the method of specimen preparation has no effect on the results. The identification made by Ohara (10) and by Horio and Kondo (11), that the site of the preferential acid dyeing was the portion of the fiber not dyed preferentially by basic dyes, was based on their observation that the' outer, convex portion of the crimp curvature was preferentially stained by basic dyes, and the inner, concave portion by acid dyes. The work of Dusenbury and Coe (2) indicates this result to have been an exceptional one over a wide range of dyeing conditions, acid dyes, like basic dyes, stain the outer, convex portion of the crimp curvature. This has been observed when dyeings were made on bulk samples of 70's wool (Australian Merino), as well as on a sample of 64's wool (U.S. Rambouillet). It was found possible in one instance to reproduce Horio and Kondo's observation, using their dyestuff (Ponceau 2R, C. I. 79) and rather drastic dyeing conditions: thirty minutes at the boil in a phosphate buffer solution of pH 7.0. Further observations on these fibers that had the inner portion of the crimp curvature preferentially stained showed that the boiling buffer solution had reduced crimp frequency and had actually reversed the crimp (presumably by differential supercontraction), placing the orthocortex fraction on the inner rather than the outer portion of the crimp curvature. The preferentially dyed portion of the fiber was identified as ortho by its preferential lability to alkali. This is a possible explanation of the Horio and Kondo results with Ponceau 2R. It does not explain why they ob- served it at 60øC., whereas Dusenbury and Coe observed it only at the boil. The observed dyeing behavior shows that the orthocortex is preferentially

EFFECT OF BILATERAL STRUCTURE ON KERATIN FIBERS 36l stained by both acid and basic dyes and that the phenomenon is to be associated with the greater reactivity or accessibility of the orthocortex to chemical reagents rather than to differences in the number of sites for dyeing in the two portions of the wool fiber cortex. Such a finding is also consistent with the fact that all keratin fibers, regardless of their origin, exhibit about the same acid-combining capacity. Another point raised by this finding that the orthocortex is preferentially stained by both acid and basic dyes is its possible relationship to objections raised by Harris (6) in respect to the amino acid analyses of Golden, Whitwell and Mercer (5). The paracortex-plus-epicuticle fractions analyzed by Golden et al., were prepared by the previously described process of supercontraction followed by enzymatic digestion by microscopic observation this process appeared to extract thl•eorthocortex cleanly, leaving behind a paracortex-plus-epicu- ticle fraction. Harris has pointed out that, if this preparative procedure were to extract residues preferentially from the paracortex containing lower amounts of cystine and of basic amino acids such as arginine, the remaining paracortex would contain artificially high amounts of cystine and basic amino acids. At present no direct method has been used to determine the cystine contents of the two portions of the wool fiber cortex. The cystine differences observed, however, are large and in general are in accord with the cystine contents found by a direct method for other keratin fibers that are primarily "ortho" or "para" in nature. These are discussed later in this paper. No such indirect confirmation of the amino acid analyses is avail- able for the observation of the excess of basic amino acids in the para- cortex. The confirmation given to this observation by the differential dyeing results of Ohara and of Horio and Kondo appears now to be spurious. In fact, a recent study of the amino acid composition of •-ACTH (a physiologically active component of corticotropin from hog anterior pituitary) by Bell and others of the American Cyanamid Company (1) indicates a basis for the objections of Harris. They found this protein (molecular weight 4566) to consist of 39 amino acid "toohomers," of which 16 were different, to have its acidic and basic amino acid components grouped more or less separately from one another along the molecule, and to have its acidic amino acid components preferentially extracted when treated with enzymes. To the extent that an analogy may be drawn between the work of Golden et al. (5), and these findings, that enzymes can extract dicarboxylic amino acids from a complex protein under some circumstances, any assumptions made regarding the absence of such extraction may be incorrect. Still another indication of ortho-para differentiation in wool fibers is observed when a wool fiber is first subjected to acid and then exposed to alkali (3). A digression is in order at this point. In connection with the wool research programs at Textile Research Institute, an investigation