496 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS All of these enzymes were commercially available and their sources of origin with their claimed activity are shown below: Table II Enzyme Declared activity Manufacturers BP Novo 1.24 Artson units/G Novo Industri Trypsin 25.60 Anson units/G Novo Industri Prozyme -- Amano Pharm Co. Ltd Papain Conforms to BPC 1954 Koch Light Lab. Ltd Papain W.S. 60 000 units/G E. Merck Ficin 3 900 units/G E. Merck Lioease MY 30 000 units/G Meito Sangyo Co. Ltd The reactions between the enzymes and stratum corneum were carried out as follows. A 50-mg sample of stratum corneum was mixed with 10 ml of the enzyme solution in a sterile phosphate buffer at pH 6.5. The mixture was incubated at 36øC and samples taken at 1 h and 3 h. Each reaction was compared with the following blanks: (a) 10 ml of enzyme in the sterile buffer (b) the sterile buffer itself (c) the stratum corneum in the sterile buffer. Samples were taken at 1 h and 3 h. The enzymes, soluble proteins, and long- chain peptides, were precipitated with 30•o trichloroacetic acid, the solu- tions then being centrifuged for 15 rain and the supernatant liquid decanted. Two methods were then used for estimating the extent of the reactions occurring. (1) The proteolysis was estimated by a modification of the Folin/Lowry method (10), which is based on the reaction of Folin and Ciocalteu's reagent (11), at alkaline pH, with certain amino acids. The optical density (OD) of the resultant blue colouration was measured using an EEL spectrophotometer at 630 nm. Readings were taken at 1 h and 3 h and the results expressed in terms of 'activity' (Table III) where:

THE EFFECT OF ENZYMES ON STRATUM CORNEUM 497 Table III Enzyme Activity after 1 h Activity after 3 h BP Novo 0.0050 0.2400 Trypsin 0.1150 0.1700 Prozyme 0.1025 0.0050 Papain 0.0250 0.2000 Papain W.$. 0.0625 0.1150 Ficin - 0.0315 0.0867 Lipase MY 0.0050 0.0200 Ltpase MY + BP Novo 0.1250 0.1300 Activity-- OD of Test - (OD of substrate blank + OD of enzyme blank). A standard protein/optical density curve for the Folin/Lowry estimation using crystalline bovine albumin was constructed from which Table IV was prepared: Table IV Solubilized proteins expressed as [tg/ml of Albumin Enzyme 1 h 3 h BP Novo 1.3 78.2 Trypsin 37.5 55.3 Prozyme 33.2 1.3 Papain 8.0 65.0 Papain W.S. 21.0 37.5 Ficin -- 28.0 Lipase 1.3 6.2 Lipase + BP Novo 40.5 42.3 (2) The keratinolysis was determined by estimation of the free protein sulphydryl groups using the method of Boyer (12). This method utilizes the formation of a mercaptide derivative using a p-chloromercuribenzoic acid with L-cysteine HC1 being used as a standard source of sulphydryl (--SI-I) groups. A standard pattern of dilutions was made as shown in Table V: