RED PIGMENTARY SYSTEM 79 acids may be absent, because cysteine and dopa are required for the synthesis of the chromophore. The presence of iron as an essential com- ponent of the red pigment-protein complex has been challenged (3) on the ground that the chromophore itself contained no iron. A similar argument advanced in the case of hemoglobin would deny its iron-pro- tein nature, because the porphyrin nucleus is free of iron. Nevertheless, the possibility that iron may be a contaminant, chelated by the chromo- phore in the course of the purification of the pigment, must be considered. Three types of experiments were performed to test this possibility: 1. The pigments were isolated under mildest possible conditions with careful avoidance of external contamination which might lead to iron uptake (splitting of PP and siderins with KSCN, followed by dialysis •vithout gel filtration). Such fractions still contained traces of protein (3-5%) with the enrichment of iron (to about 1.5%) observed earlier by ourselves (2) and Boldt (5). 2. We found that PP and siderins alike could be split with a variety of iron-binding anions (thiocyanate, ferrocyanide, cyanide, azide, fi-hy- droxamate) to a nondialyzable and dialyzable fraction. The latter dis- played the typical properties of the chromophore unless a secondary re- action occurred, as with cyanide. 3. We observed that ferric ion, when added to the chromophore and chromophore peptide fractions of the siderins, abolished the color and absorption spectra of these compounds. After addition of ferric ions this change could be reversed with acids, as in other ferric-phenolic com- plexes (higher concentrations of iron irreversibly altered the chromo- phore). Thus, it is unlikely that combination of the chromophore with iron would occur in the course of purification. All these observations give added support to the structure: protein- Fe(III)--(cysteinyldopa)2-derivative. By analyzing the peptides still at- tached to the chromophore in the incompletely purified iron-rich frac- tions, information may be obtained about the mode of attachment of iron to the protein molecule. Preliminary data suggest a marked enrichment in glycine. IMPLICATIONS OF THE CHROMOPHORE STRUCTURE Chemical identification of the chromophore accounts for many unique properties of the red pigments. These are summarized briefly. 1. As cysteine is incorporated in the chromophore, the sulfur cannot form the thioether bridge which in the black melanins binds them to the

80 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS protein part of the melanoprotein molecule. The protein part of the red pigment is very loosely attached and can be removed by column chromatography, gel filtration, or splitting with iron-complexing ions. This easy operation opens up new avenues for studies of malignant mela- nomas. 2. The chromophore of the red pigment has a much smaller molecu- lar weight (561) than has been estimated for the black melanins (in the tens of thousands). It is less polymerized and forms a less firm molecular aggregate with its protein portion. Electron microscopic pictures of "red melanosomes" (which should be called erythrosomes) reflect this circumstance. They are not as fully "melanized" as their black counter- parts, but instead form a loose, regularly patterned network of pigment. Their ovoid rather than round shape has been attributed to the lesser stress exerted on their membranes by the incompletely polymerized red pigment (12). 3. The first step in pigment formation, the conversion of tyrosine to dopa, is the same in red and black pigments. Therefore, both red and black melanocytes give a histochemical and chemical tyrosinase reaction. 4. A major consequence of this common first step is the potentiality of red pigment cells to switch to black melanin production conversely, rerouting of black melanogenesis toward the synthesis of red pigments also may occur. The possible interconversion of these two pathways will be discussed below. INTERCONVERSION BETWEEN RED AND BLACK PIGMENT FORMATION A simplified scheme of a possible switch mechanism between the red and black pigmentary systems is as follows (6): tyrosine --Y dopa • dopa-quinone • black melanins cysteine J• cysteinyldopa • red pigments It is possible to divert dopa toward the black pathway by decreasing the "side-tracking" effect of cysteine conversely, the black system may be forced to produce red pigments through an "overdose" of cysteine. Con- versions of black pigment forming systems to red pigment forming sys- tems (and vice versa) are known to occur under experimental or physio- logic conditions. Starvation in certain black fowls leads to the production of red leathers. It has been postulated that during starvation the decreased growth rate of feathers prolongs the exposure of the feather matrix to a