Spectral absorption and fluorescence of coproporphyrin isomers I and III and the melting-points of their methyl esters.

Both the determination and the identification of the naturally occurring coproporphyrin isomers I and III depend upon the exactness of the data relating to their physical properties and to those of their methyl esters. Quantitative estimation of these isomers involves spectrophotometric or fluorimetric comparisons with standard solutions usually prepared from the crystalline tetramethyl ester of isomer I. The accuracy of such determinations depends upon the purity of this ester and also upon the knowledge of the spectral absorption of the isomers or of the relative fluorescence efficiencies of the two molecules. Differential identification of the coproporphyrin isomers is achieved through differences in the properties of their tetramethyl esters, particularly their solubility in organic solvents and their melting-points. A clean-cut separation of the esters is not easily effected. This difficulty must be borne in mind in considering the melting-points of the methyl esters isolated from natural sources, unless careful recrystallizations have been done. In studies of porphyrin metabolism, an obvious essential is the definition of those properties which form the basis of quantitative methods of estimation and of identification. This paper presents data obtained in an attempt to correlate the spectral absorption and fluorescence of the isomers and the m.p.'s of their esters, so that their additive and constitutive properties may be more closely defined. The necessity for such data became apparent when we found that solutions prepared from synthetic coproporphyrin I tetramethyl ester (as received from Prof. H. Fischer) gave extinction coefficients, E1 J(wlv), as much as 12% lower than those made up from the same specimen after recrystallization. Recrystallization, however, caused no appreciable change in m.p.'s, which were throughout within the generally accepted range of good values for this substance (Table 1 and Fig. 4). Similar observations were made on synthetic coproporphyrin III tetramethyl ester and on several natural specimens. This experience led us to investigate the criteria for the purity of the tetramethyl esters of coproporphyrins I and III, and to attempt to define more closely the additive properties of standard solutions of coproporphyrins I and III. EXPERIMENTAL