338 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS TABLE 2--EFFECT OF THE EXTRACTION PROCEDURE ON THE MOBILrrv OF THE Nos-FIBROUS PROTEIN OF BEEF SNOUT EPIDERMIS ----Extraction Procedure and Treatment-- --- Mobility - • Molar Tern- (cm. 9' volt -• Concen- pera- sec. -• X 10 a) tration Sample ture, Time, Descending of Urea No. øC. Days Nature of Extract Limb 10 1 0 10 One extract of epidermis 3.3 6 2 20 67 Proteins ofisoelectric point 5.0 of pH 6.3 treated with a 6 M urea solution 6 3 0 7 Supernatant after dialysis 2.9 6 4: 20 22 Clot from dialysis (3) **, re- 3.1 homogenized in 6 M urea 2 5 0 7 First extract of epidermis 3.8 2 6 0 7 Third extract of epidermis 3.1 (5)** 2 7 0 7 From purification of fi- 5.1 brous protein obtained as a gel-like mass* 1 8 0 7 One extract of epidermis* 3.4: 0.5 9 0 7 One extract of epidermis 3.6 0.5 10 0 7 One extract of epidermis* 3.4,4.8,t7.4t * Final solution of non-fibrous protein centrifuged for 5 hr. at 130,050 X gravity. ** These numbers refer to the Sample No. (column 2). •' Mobility of a substance present in small amounts. The mobilities of the non-fibrous protein also appear to be independent of the concentration of urea employed for its extraction (Table 2), except for the pH 4.5 protein, isolated from the insoluble proteins of isoelectric point of pH 6.3 by the action of urea, which had a mobility of 5 cm. 2 volt -• sec. -I X 10 -5. Evidently the protracted action of urea on the insoluble proteins of isoelectric point of pH 6.3 had considerable effect upon its fibrous and non-fibrous components. Finally one of the non-fibrous proteins which was obtained from the purification of one of the fibrous proteins (2.0 M urea) had a mobility of 5.1 cm. •' volt -1 sec. -1 X 10 -5. One of the fibrous and non-fibrous proteins prepared from the 0.5 M urea epidermal extracts showed more peaks electrophoretically than were found for these proteins isolated from the more concentrated urea extracts. The fairly symmetrical electrophoretic patterns of some of the fibrous and non-fibrous protein solu- tions does not necessarily imply homogeneity, but they do indicate that the procedures employed for the purification of these proteins are quite effective. Investigations are under way employing sedimentation analysis and paper electrophoresis to obtain more information on the homogeneity and properties of the fibrous and non-fibrous proteins. When beef snout epidermis is placed into a 6 M urea solution, extraction of the proteins occurs first in the lower layers of the Malpighian stratum according to Rudall (17) and to our histological observations (Figs. 1 to 5). Dilute urea solutions (0.5, 1.0 and 2.0 M) did not extract from the beef snout

PROTEINS OF EPIDERMIS, RELATION TO AGING SKIN 339 epidermis proteins which formed a clot upon dialysis of the extracts against distilled water. These concentrations of urea probably extract most of the structural proteins from the lower strata of the epidermis. Six or 10 M urea solutions apparently extracts from epidermis more complex protein mate- rial since dialysis of these epidermal extracts results in a clot. The latter is cleaved by 6 M urea solution to give the fibrous and non-fibrous proteins. According to Rudall these proteins are chemically bound to form a complex protein which comprises the clot (17). The proteins of the clot and some of the fibrous and non-fibrous proteins in all probability are extracted from the upper Malpighian layers of the epidermis where the keratinization proc- ess is nearing completion and consequently more complex proteins are present. The insoluble proteins of isoelectric point of pH 6.3 apparently form the clot material since this insoluble protein is further split by a 6 M urea solution to yield proteins of isoelectric points of pH 4.5 and 5.5. Since the yields and the type of the proteins obtained from the 6 M urea extract of epidermis are respectively greater and different (Chart I) than those isolated from the 2 M urea extract (Chart III), it seems unlikely that the dilute and concentrated urea solutions extract proteins of the same com- position from this tissue. The use of micro-methods for the study of the structural proteins may permit the isolation of them from the lower, inter- mediate and upper strata of the epidermis by the procedure employed by Rudall (17). Proteins were also extracted with a 6 M urea solution from hyperplastic (methylcholanthrene and benzpyrene induced) mouse epidermis. Dialysis of the extracts of this tissue against distilled water resulted in a ftocculent precipitate with little tendency to form a clot. Complete fiocculation of the very insoluble proteins, part of which came down upon dialysis, oc- curred at pH 6.2 to 6.3. The supernatant fraction which resulted from the separation of the insoluble proteins by centrifugation (24) gave the fibrous and non-fibrous proteins. The fibrous protein had a mobility of 2.5 cm. 2 volt -• sec.-• X 10 -5 which is lower than that obtained for the same protein of beef snout epidermis. The non-fibrous protein had a mobility of 3.2 cm. 2 volt -• sec. -• X 10 -5 which is the same order of magnitude as that found for this protein of beef snout epidermis. The process of keratinization in epidermis is not well understood al- though some attempts have been made to postulate the mechanism of the biosynthesis of keratin. Mercer has proposed that in the intermediate zone of the Malpighian layer of the epidermis the molecules of the primary pre-keratin precursors of unknown shape or size may exist as organized particles of a corpuscular type protein (14). End-to-end linkage of the latter in the upper Malpighian stratum brings about formation of a fibrous form of the protein. Since the latter, in contrast to the hardened keratin, is digested with trypsin and two N hydrochloric acid and is converted