CELL MEMBRANE COMPLEX 455 Since the cell membrane lipids of cortical cells are not bound by thioester linkages as in cuticle cells, but are bound by polar bonding and ionic bonds, a high level of cysteine links via an UHSP is not necessary for the cell membrane proteins of cortical cells. The resistant membrane material from the reaction of performic acid on wool fi ber by Bradbury et al. (71) provides only about 62% of the amount of cystine as the composition of the Allworden membrane by Allen et al. (30) and is consistent with the expectation of a lower cystine content in cortical cell membranes versus cuticle cell membranes. In ad- dition, one would expect the proteins of the cortical cell membranes to contain a larger number of basic amino acids (for bonding to cholesterol sulfate and to fatty acids), and more polar sites such as carboxyl groups would be preferred for polar bonding to fatty acid groups of lipids we fi nd nearly twice the basic amino acid content for salt link formation to cholesterol sulfate and fatty acids and more carboxyl groups of acidic amino acids in the membranes by Bradbury et al. (71). Furthermore, one would expect this performic acid-derived membrane material by Bradbury et al. to be richer in cortical cell membranes than cuticle cell membranes, since the cortical cell membranes should be a higher percentage of the total membrane matter in total wool fi ber, and the data is cer- tainly consistent with this expectation. A cleaner experimental scheme to isolate pure cortical cell membranes would be to start with pure cortex to exclude cuticle cell membranes and A-layer proteins. Pure cortex from human hair could be provided by the glass fi ber method of Wortmann et al. (74) and then perhaps by the performic acid reaction or another scheme to provide cortical cell membranes in the absence of cuticle contamination for further workup and analysis. PROTEINS EXTRACTED FROM HAIR/WOOL BELIEVED TO BE FROM THE DELTA LAYER OF THE CMC Leeder and Marshall (51) extracted Merino wool with formic acid and also with n-propanol/ water (50/50). Proteinaceous matter was removed from the hair fi bers with each of these solvent systems. With formic acid, these scientists concluded that the proteins were at least partially derived from the CMC, most likely from the delta layer because the extract contained virtually no cystine. If this proteinaceous material is from the delta layer, it most likely is not from the central proteins sometimes called the contact zone (or it con- tains small amounts of these proteins) because Naito et al. (75) have provided evidence that the contact zone contains hydrophilic protein with disulfi de bonding. Leeder and Marshall (51) concluded that the proteins derived by their own propanol/ water extraction of wool is not entirely from the cell membrane complex, but that they also contain high glycine–tyrosine proteins possibly derived from the cortex. The amino acid composition of proteins extracted by formic acid, by n-propanol/water, and by chlo- roform/methanol are compared with that of the Allworden membrane in Table V. Logan et al. (56) have shown that a chloroform–methanol azeotropic mixture provides a very different mixture of proteins than the high temperature propanol/water extraction (see Table V). Could this chloroform–methanol extraction be partially derived from corti- cal cell membranes or part of the outer lamella (outermost layer) of the delta layer pro- teins of the CMC? Since Mansour and Jones (37) have shown that chloroform/methanol extraction provides large changes to the cortex–cortex CMC in wool, it is possible that these proteins removed by chloroform/methanol extraction are at least partially attached to beta layers and are at least in part delta layer proteins of the cortex–cortex CMC.

JOURNAL OF COSMETIC SCIENCE 456 The method of Swift and Holmes (6) has been used by several different researchers to obtain proteinaceous matter believed to be partially derived from the CMC. This method involves dissolving matter from hair using papain with a reducing agent such as bisulfi te or dithiothreitol (DTT). Bryson (76) conducted a series of experiments from which he concluded that the laminated structure observed under the TEM following a 72-hour digestion of wool fi bers with papain and reducing solution (somewhat standard proce- dure) is not derived entirely from the CMC. Prolonging the digestion beyond 72 hours increased the number of laminated layers beyond what could be accounted for by the number of cortical cells in a fi ber cross section. Bryson concluded that the CMC lipids were rearranging with other proteins and peptides to form these laminated layers. Mass spectrometric analysis of the proteins of the digestion residue indicated that the majority of the protein component was papain, suggesting that the CMC lipids had rear- ranged with papain to form the laminated structures. Therefore, Bryson concluded that it is not possible to isolate pure proteinaceous CMC by papain digestion. These conclu- sions by Bryson are consistent with those of Swift and Bews (77), who concluded that although treatments of keratin fi bers with enzymes and reducing agents do cause separa- tion of cells, they could fi nd no evidence of dissolution of the cuticle CMC via critical electron microscopic examination of treated hair sections. Therefore, the value of this method for isolation of CMC proteins is limited because of contamination with papain. To repeat, the diffi culty in isolating pure cell membrane proteins and pure delta layer proteins that are free of extraneous proteins and proteins from other regions of the fi ber is the primary obstacle to our understanding of the composition and structures of the proteins of this important region of the fi ber and the reason that the composition of the CMC proteins is still not adequately characterized. Table V Proteins Extracted from Wool with Formic Acid and n-Propanol/Water Compared with the Allworden Membrane Amino acids Allworden membrane (30) Formic acid (50) n-propanol (51) CHCl3/MeOH (56) Asp 3 5.7 3.7 6.2 Glu 8.6 7.2 2.4 7.5 Thr 2.1 3.8 3.2 5.1 Ser 14.3 8.1 11.7 13.3 Tyr 0 12 16.4 3 Pro 4.2 4 5.2 6.4 Gly 23.8 19.2 25 11.9 Ala 3.2 5.2 2.2 8 Val 5.6 4.2 2.8 5.9 Ile 1.2 3.3 0.8 3.5 Leu 2.9 9.2 6.1 8.1 Trp Phe 0.4 5.2 7.8 3.6 His 0.2 1.2 0.7 1 Lys 4.5 4 0.8 2.7 Arg 2.5 6.2 5.2 4.1 Met 0 0.9 0.2 0.8 Cys 21.1 0.4 5.5 9 Totals 97.6 99.8 99.7 100.1