PROTEINS OF EPIDERMIS, RELATION TO AGING SKIN 341 lated from the 2 and 6 M urea extracts formed milky suspensions respec- tively in 2 and 6 M urea solutions. The protein then became soluble fol- lowing further disaggregation by the urea. Since the polydispersity of each molecular species is considerable in urea extracts of cow lip and snout, according to Mercer and Olofsson (23), differences in the state of aggrega- tion of the fibrous protein may not be detected by electrophoresis. The behavior of the epidermal extracts and of the fibrous proteins obtained therefrom as a function of the concentration of urea suggest possibilities of the isolation of some of the units involved in the synthesis of keratin. In the epidermis of man there appears to be no distinct morphological alteration with age which would suggest possible chemical changes in this tissue. The biosynthesis of' keratin in its step-wise fashion allows for pos- sibilities or irregularities in this process. For example, the monomers of the structural proteins of feather keratin have a great tendency to associate by crogs linkages through sulfhydryl groups to form complex proteins ac- cording to Woodin (29), and our studies show that the fibrous protein of epidermis possesses this same property. The manner, enzymatic or by other means, by which cross links are selectively broken or synthesized and thus reduce or increase respectively the molecular weight of the cross linked aggregates might prove to be of great significance for an under- standing and control of changes in protein systems that are associated with the process of aging (30). There appears to be similarities between the effects of cross linking on proteins and the changes which proteins undergo in the aging organism (30). These include a reduction in the ability to re- tain bound water, loss of elasticity and reduced solubility or peptizability. Alterations in cross linkages, such as might occur in a steric position, or of a chemical nature that prevents some or a few of the usual associations through sulfhydryl groups might lead to a keratin with less desirable elastic or pliable characteristics. Actually association of the keratin precursors apparently can take place in the outer most levels of the epidermis, since in these strata sulfhydryl as well as disulfide groups are present according to Van Scott and Flesch (31). The high urea content of epidermis (32, 33) may play some role in maintaining the structural proteins in a less highly aggregated state, but the influence of age on the urea content of aging skin has not been investigated. SUMMARY 1. The effect of single and successive extractions with urea of various concentrations on the yields and mobilities of the fibrous and non-fibrous (structural) proteins of beef snout epidermis was investigated. 2. The extraction of beef snout epidermis once with 0.5, 1 or 2 M urea solutions or successively with three extractions of 6 and 2 M urea solutions had little e•i•ct upon the mobility of the fibrous and non-fibrous proteins

342 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS at pH 7.0 in phosphate buffer. The amount of both proteins obtained by the successive extraction procedure decreased with each successive extrac- tion. The second and third extraction of the epidermis with a 6 M urea solution yielded only the fibrous protein, whereas these extractions with a 2 M urea solution gave both proteins. 3. Six or 10 M urea solutions gave extracts of beef snout epider- mis which yielded clots following dialysis of the extracts against distilled water. Clots were not formed following dialysis of 0.5, 1.0 and 2.0 M urea extracts of epidermis against distilled water. The concentrated urea solu- tions apparently extract more complex keratin precursors l¾om the upper strata of the epidermis. 4. The protein material of the clots which was obtained following dialy- sis of the 6 and 10 M urea epidermal extracts against distilled water and the insoluble proteins of isoelectric point of pH 6.3 which was acquired fol- lowing acidification of the dialysis sac contents appear to be of somewhat similar composition. This is so because they were both cleaved by 6 NI'urea solutions to give the fibrous and non-fibrous proteins. The mobility of the proteins obtained from the insoluble proteins of isoelectric point of pH 6.3 was significantly higher than that obtained by direct extraction of the epi- dermis or from the clot. 5. Histological examination of beef snout epidermis left in 6 M urea solutions for periods from one to twenty-four hours revealed that this solu- tion dissolved, along with other cellular components, the tonofibrils. The granular layer of the epidermis and the keratin coat appeared to be intact histologically. 6. Possible changes in the structural proteins of epidermis with age are briefly discussed. REFERENCES (1) Cowdry, E. V., Cooper, Z. K., and Smith, W., "Program of Research on Ageing of the Skin," f. GerontoA, 2, 31 (1947). (2) Cooper, Z. K., and Schiff, A., "Mitotic Rhythm in Human Epidermis," Pro½. So½• Exp. Biol. Med., 39, 323 (1938). (3) Cooper, Z. K., "Mitotic Rhythm in Human Epidermis," a•. Investigative Dermatol., 2, 259 (1939). (4) Thuringer, J. M., "Studies on Cell Division in the Human Epidermis," .4nat. Record, 40, 1 (1928). (5) Cowdry, E. V., and Thompson, H. C., Jr., "Localization of Maximum Cell Division in Epidermis," Ibid., 88, 403 (1944). (6) Cooper, Z. K., "Ageing of Skin," Cowdry's Problems of Ageing, Third Edition. Balti- more, The Williams and Wilkins Company (1952), p. 764. (7) Evans, R., Cowdry, E. V., and Neilson, P. E., "Ageing of Human Skin. I. Influence of Dermal Shrinkage on Appearance of the Epidermis in Young and Old Fixed Tissue," .4nat. Record, 86, 545 (1943). (8) Ejiri, I., "Studien fiber die Histologie der menschlichen Haut. III," a•. Dermatol. Urol. [•apan], 41, 8 (1937). (9) Hill, R., and Montgomery, H., "Regional Changes and Changes Caused by Age in the Normal Skin. A Histologic Study," a•. Investigative Dermatol., 3, 231 (1940). (10) Thuringer, J. M., and Cooper, Z. K., "The Mitotic Index of the Human Epidermis, the Site of Maximum Cell Proliferation and the Development of the Epidermal Pattern," •lnat. Record, 106, 89 (1950).