THE PIGMENT MELANIN OF THE SKIN AND HAIR 35 alanine and the normal formation of pigment. There is no reaction to dioxyphenylalanine in albinotic skin and vitiligo, while an increase re- action is a necessary accompani- ment and the first evidence of all processes of pigment formation Bloch (7), Peck (14), Miescher (8). Further pigment is the demonstra- tion of pyrocatechol, the natural deaminization product of dioxy- phenylalanine, in the urine of pa- tients with generalized melano carcinoma. One cannot refute the assumption of th.e propigment function of di- .oxyphenylalanine on the ground that its presence has not been demonstrated in the animal body. The deeper understanding of inter- locking chemical processes, such as alcoholic fermentation, has shown that an intermediary substance could easily escape analytic detec- tion and yet form a pivotal phase in the course of the enzymatic process. A great many other criticisms have been raised against Bloch's assumption of a specific dioxy- phenylalanine oxidase. They have been based mainly on the fact that there are other catalytic systems of wider distribution that convert di- oxyphenylalanine into melanin. Polyphenolase, an enzyme of almost ubiquitous occurrence in living mat- ter, acts on polyphenol derivatives, especially orthodiphenol (pyrocate- chol) and its derivatives, such as epinephrine, dioxyphenylalanine, etc., an oxidation that in the pres- ence of amind groups leads to the formation of melanin. The effects of this enzyme are often overshadowed by a concomi- tant heat-stable and therefore non- enzymatic mechanism of oxidation, which is attributed to the iron sys- tem of the cell. The differentiation between these two catalysts has to be based on careful quantitative studies before one is entitled to re- fute the enzymatic character of such reactions. Tyrosinase is another enzyme that can convert dioxyphenylalanine into a melanin. It is found in insects, fungi and higher plants such as po- tatoes. The mechanism, of the formation of melanin from tyrosine by this enzyme has recently been elucidated by Raper (15). He showed that the first step in the oxi- dation of tyrosine is the introduction of a second hydroxy group into the benzene ring yielding ,3-4 dioxy- phenylalanine. The later stages of formation of melanin are due to a less specific mechanism the enzy- matic nature of which is doubtful. Tyrosinase also acts on other mono- phenol derivatives such as para- cresol, tyramine, etc. Unless the enzyme is purified, it also oxidizes the substances mentioned because of contamination with polyphenolase. There is no evidence of the identity of either tyrosinase or polyphen.olase with dioxyphenylalanine oxidase. The oxidation of dioxyphenylalanine by tyrosinase can certainly riot be used as evidence for the identifica- tion of this enzyme with the pigment oxidase, since the outstanding fact remains that the dioxyphenylalanine oxidase acts only on dioxyphenyl-

36 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS alanine and not on tyrosine (Bloch and Schaaf (7) and our own experi- .ments). The polyphenol oxidase of the myelogenic cells acts on many poly- phenol derivatives and easily oxi- dizable substances such as epi- nephrine, pyrogallol and phenylene diamine (Bloch and Peck (1{5)) as well as on dioxyphenylalanine. In view of the higher specificity of the dioxyphenylalanine oxidase of the melanoblasts, any theory attempt- ing to identify dioxyphenylalanine oxidase with polyphenolase (Oppen- heimer (17)) is just as untenable as that of the identity of dioxyphenyl- alanine oxidase with tyrosinase. There are a number of proofs for the existence of an individual di- oxyphenylalanine enzyme: (1) The dioxyphenylalanine oxidase is ex- tremely labile against the destruc- tive influences of heat and especially of certain poisons as compared to the less specific, more rugged poly- phenolase and the nonenzymatic oxidizing catalysts of the cell. (2) Its action is confined to a much narrower hydrogen ion concentra- tion than that of either polypheno- lase or tyrosinase. (3) As demon- strated by Bloch and Schaaf (7), the dioxyphenylalanine oxidase is so specific as to be inac'tive against a substrate showing the slightest change in the chemical constitution. Recently Mulzer and Schmalfuss (18) cast doubt on the specificity of the reaction to dioxyphenylalanine because in their hands 3-4 dioxy- phenylethylamine (oxytramine) also gave a positive product of dioxy- phenylalanine, it seems that this finding rather corroborates Bloch's theory of the formation of pigment. In the following series of experi- ments new evidence is presented for the specificity of the reaction to dioxyphenylalanine. The optical specificity of the action of enzymes has been studied thoroughly in several groups of hydrolyzingenzymes. The problem of optical specificity in oxidizing enzymes has been investigated with tyrosinase. According to Abderhalden and Guggenheim (19), this enzyme is optically selective to a limited degree only, showing some preference for the leveroratory natu- ral substrate. We (Peck, Sobotka and Kahn (20)) investigated the optical speci- ficity of the dioxyphenylalanine oxidase, and we found that it was strictly specific for the levorotatory natural form of dioxyphenylalanine (21). The dextrorotatory antipode was left untouched by the enzyme (22). Bloch and Schaaf (7) have independently made the same ob- servation. The formation of melanin from dioxyphenylalanine by crude potato tyrosinase (prepared according to Lichtman and Sobotka (23)) is rapid. It takes place to a con- siderable degree even when the true tyrosinase is destroyed by heat. The potato preparation acts equally on dextrorotatory and on levo- rotatory dioxyphenylalanine, as is to be expected in a material rich in polyphenol-oxidizi.ne catalysts. The enzymes responsible for the