PROTEINS OF EPIDERMIS, RELATION TO AGING SKIN 327 present in the epidermal structures of reptiles and birds in addition to the a-form (20). Another important consideration of the structural proteins of epidermis aside from their role as the precursors of keratin, is their close association with or as an integral part of the tonofibrils or intracellular bridges. This association was emphasized by Giroud and Champetier (21) and has been further strengthened (22) for both the Malpighian and keratinized layers of epidermis. The tonofibrils are found close to the cell walls rather than uniformly dispersed throughout the cytoplasm, and they are perpendicular to the skin surface in the stratum germinativum, but become more parallel to the surface as the cells move outward. Giroud and Champetier con- sidered that the continuity of the tonofibrils may help in holding the squa- mous cells together and thus explain their resistance to rupture (21). This resistance to rupture may be a consequence of the ability of the keratin proteins on the tonofibrils to elongate instead of breaking. From the results discussed above, it may be •concluded that a protein or proteins with the keratin x-ray diffraction pattern (a-form) exists in the Malpighian layer of epidermis. These keratin precursors apparently be- come the true keratin between the stratum granulosum and stratum cor- neum (14, 23). This concept was confirmed by Rudall who has made the most significant contributions to our knowledge of the structural proteins of epidermis (17). He employed cow snout epidermis which is some 1.5 min. thick, is practically hair free and is low in lipids (17). Rudall was able to extract from cow snout epidermis with a 6 M urea solution a fibrous pro- tein which he called epidermin. Films of this protein showed an a-type diffraction pattern which changed to the/g-type upon stretching. A non- fibrous protein was also extractable with a 6 M urea solution and films of this protein gave the/g-type diffraction pattern. Both of these proteins were partially purified by dialysis of the urea extractable material from epidermis this procedure was followed by resolution of the fibrous protein in a 6 M urea solution and then after dialysis reprecipitation at the isoelec- tric point of pH 5.5. The non-fibrous protein which is more soluble in water, was partially purified by resolution in water at pH 7 and then by reprecipitation at the isoelectric point of pH 4.5. An interesting aspect of Rudall's work is that the outer, inner and middle levels of the Malipighian layer contain both proteins. The sulphur con- tent of the non-fibrous protein increases from the inner to the outer strata of the epidermis, whereas that of the fibrous protein decreases. Extensive investigations were carried out by Rudall on the x-ray diffraction patterns of the fibrous protein in its a and/3 forms and on thermal contraction of this protein and of the various strata of epidermis. Mercer and Olofsson have shown that urea extracts of cow lip and snout gave several peaks when sedimented in the ultracentrifuge (23). They

328 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS found that the sedimentation velocities showed a wide range, with a main group with S -- 1.3-1.6 sveds. From the diffusion coefficient, a molecular weight of 60,000 for this main group ofprote, ins was calculated. A frictional ratio of 3.5 was found which indicates a highly asymmetric molecule. Carruthers, Woernley, Baumler and Kress have studied by electrophore- sis some of the properties of the fibrous and non-fibrous proteins of the epi- dermis of beef snout and the mouse (24). The procedures employed for the extraction of these proteins were essentially those of Rudall. The following results were obtained: The mobilities of the fibrous and non- fibrous proteins isolated from a 6 M urea extract of epidermis at 0øC., were lower than those obtained for these proteins extracted with the same solu- tion at 28øC., or with a 10 M urea solution at the same temperature. An insoluble protein of isoelectric point of pH 6.3 was also isolated from the 6 M and 10 M urea extracts of beef snout epidermis. This protein was cleaved further by 6 M urea solution to yield proteins of isoelectric points of pH 4.5 and 5.5. Solutions of several detergents also extracted from the epidermis proteins which were unrelated to those isolated from the concen- trated urea extracts of this tissue. Binding of the fibrous and non-fibrous proteins with the detergent, sodium lauryl sulfate, to form several distinct protein detergent complexes was demonstrated by electrophoresis. In this report investigations were undertaken to determine the effect of dilute and concentrated urea solutions on the extractability of the fibrous and non-fibrous proteins from epidermis. This approach was pursued in order to ascertain whether some of the properties of the keratin precursors was a function of the concentration of urea employed for the extraction of these proteins. METHODS The procedures employed for the extraction of the structural proteins from epidermis with 6 M urea solutions were those of Rudall (17) with some modifications (24). Further experiments were undertaken to obtain more information on the effect of single and repeated extractions of isolated epi- dermis (25) with dilute and concentrated urea solutions on the yields and electrophoretic mobilities of the fibrous and non-fibrous proteins. A typi- cal extraction of beef snout epidermis with a 6 M urea solution is shown in Chart I. The supernatant fraction from the dialyzed extract after acidifi- cation with hydrochloric acid gave a small amount of protein at pH 6.3. The fibrous protein of isoelectric point ofpH 5.5 was purified as previously described (17, 24) and in addition by centrifugation at 130,050 X gravity for five hours in a Spinco Ultracentrifuge. This procedure gave rise to a small amount of a gel-like mass and a slightly yellowish supernatant frac- tion. The former in solution at pH 7.0 and the latter flocculated at pH 5.5 upon the addition of hydrochloric acid. Further treatment of the clot