Studies in enzyme cytochemistry. III. Relationships between solubility, molecular association and structure in indigoid dyes.

The bearing of lipid solubilities and substantivities of dyes on the results of cytochemical staining reactions is briefly discussed. Solubilities of indigoid dyes in various lipids are qualitatively similar to those in symtetrachloroethane. Quantitative determinations made in this solvent indicate that, in general, indigo and its 5:5'and 6:6'-substituted derivatives are sparingly soluble, whereas the corresponding 4:4'and 7:7'-substituted dyes are relatively very soluble. The enhanced solubilities of the latter dyes are consistent with the hypothesis that the substituents interfere with edge-to-edge hydrogen-bonded association otherwise present in indigo and its 5:5'and 6:6'-substituted derivatives. This hypothesis is supported by mutually consistent results given by solubility, melting point and infra-red spectrographic studies. Indigoid dyes possessing small substituents in the 4:4'-position, such as 4:4'-dichloro-indigo, are highly substantive and therefore potentially of great value when formed in cytochemical staining reactions.