330 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS with 6 M urea yielded more proteins with isoelectric points of pH 4.5 and 5.5. A second and third extraction of the epidermal residue with 6 M urea gave only the protein of isoelectric point of pH 5.5. When a 6 or 10 molar urea solution was employed to extract the epider- mis and the contents of the dialysis sac (clot plus fluid fraction) were acidi- fied with hydrochloric acid, a large amount of a stringy, jelly-like mass precipitated at pH 6.2-6.3. The proteins of isoelectric point of pH 5.5 and 4.5 remained in solution (Chart II). When the jelly-like material was treated with a 6 M urea solution (10 ml. per gram wet weight) at room tem- perature for some time, considerable amounts of proteins of isoelectric points of pH 4.5 and 5.5 were obtained. The clot and the proteins of iso- electric point of pH 6.3 are apparently split by urea into proteins having the same isoelectric points, and the results obtained with the clot are in agreement with the results of Rudall (17). These proteins may, however, be adsorbed on the clot or on the insoluble protein mass since the yields of the fibrous and non-fibrous proteins obtained from them are rather small. Studies are under way to determine whether adsorption or cleavage is re- sponsible for the liberation of more of the fibrous and non-fibrous proteins. C•IaR•' II--PRocEr•vaE ro• 'rI-IE EXTgAOTIO•r or THE P•,o•rEx•rs •,o• EPmE•,•xs wrrI• A 10 M Urea Solution Beef snout epidermis 96.6 grams I Extracted with 400 mi. 10 M urea 10 days at 0øC. I Extract (dialyzed), then acidified to pH 6.3 Precipitate I i I I pH 6.3 P pH 5.5 P pH 4.5 P (wet wg•. 115 grams) (119 mg.) (47 rag.) Extracted with 1150 mi. 6 M urea at 20 ø 2 months, 1 week i 1 pH 6.3 pH 5.5 P pH 4.5 P (discarded) (416 mg.) (172 mg.) protein. A flow chart for the extraction of the proteins from beef snout epidermis with a 2 M urea solution is shown in Chart III. With urea of this concen- tration (or of 0.5 and 1.0 M), no clot formed in the dialysis sac following dialysis of the urea extracts against distilled water. The second and third extractions of the epidermal residue gave both proteins of isoelectric points of pH 4.5 and 5.5.

PROTEINS OF EPIDERMIS, RELATION TO AGING SKIN 331 CHART III--PROCEDURE FOR THE EXTRACTIO•q OF THE PROTEINS FROM EPIDERMIS WITH A 2 M UREA SO•.UTION Beef snout epidermis (53 grams) I I. Extracted with 400 ml. 2 M urea 7 days at 0øC. I I--Extract (dialyzed)-] I I pH 5.5 P(A) pH 4.5 P(B) (50 mg.) (52 mg.) Residue beef snout epidermis I II. Extracted with 400 mi. 2 M urea 7 days at 0øC. I [ I Residue beef snout epidermis •-Extract (dialyzed)n pH 5.5 P(A) pH 4.5 P(B) III. Extracted with 400 mi. 2 M urea 14 days at 0øC. vExtract (dialyzed) Residue beef snout epider- I 1 mis (discarded) pH 5.5 P(A) pH 4.5 P(B) (19 mg.) (22 mg.) Proteins IIA and IIB lost. Proteins lB and IIIB purified as previously described (24). For the fibrous proteins (IA and IIIA) the last centrifugation was done in a Spinco Ultra- centrifuge at 130,050 X g for 5 hr. P -- protein. A Perkin-Elmer, Model 28 electrophoresis apparatus was employed using standard techniques (27, 28). Electrophoresis was carried out in phos- phate buffer, pH 7.0 (r/2, 0.20). ... ?•½.. ,.-[•..,..., .... ,.,., : -C• ? - ..•.. ß • -• •..:.• ,: '• ,• •%•.,,., ,: • .• •:•. •'.•.• ...... •.. •....? :- -.•.• Figure 1.•Beef snout epidermis, X 80. Note the various strata this tissue.