PROTEINS OF EPIDERMIS, RELATION TO AGING SKIN 337 Beef snout epidermis extracted with 0.5 and 1.0 M urea solutions did not yield a clot in the dialysis sac following dialysis against distilled water. Figure 9 shows an electrophoretic pattern of the non-fibrous protein from a 0.5 M urea extract. The peak is quite symmetrical and it had a mobility of 3.4 cm. 2 volt -x sec. -1 ( 10 5. There were also present two other minor components with mobilities of 4.8 and 7.4 cm? volt -x sec. -1 X 10 5. The fibrous protein from one of the 0.5 M urea epidermal extracts had an dec- trophoretic pattern with three large peaks (Fig. 10) of mobilities of 3.9, 4.1 and 4.2 cm? volt -x sec. -x X 10 -5. Figure 10.--Electrophoretic pattern of the fibrous protein isolated from a 0.5 M urea extract of epidermis, descending limb. The mobilities of the fibrous protein extracted from epidermis under vari- ous conditions are given in Table 1. The mobility of this protein at pH 7.0 appears to be independent of the concentration of urea employed for its extraction. Only the mobility of the protein isolated from the urea treated proteins of isoelectric point of pH 6.3 is significantly higher (mobility of $.0 cm? volt -• sec.-• X 10 -5) than that given by this protein obtained by the other extraction procedures. The insolubility of the fibrous protein necessitated redispersion and resolution in urea several times during the purification process, and such treatment must produce a protein of different composition than is found originally present in epidermis. Other physico- chemical techniques may show some differences in the properties of the fibrous protein as a function of the urea concentration employed for its extraction.

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