The extraction and paper chromatography of hemins.

The classical procedure for the identification of hemins as prosthetic groups is to isolate the hemins, convert them into porphyrin esters, and identify the esters by their spectra and melting points. This procedure usually requires considerable amounts of material, gives poor yields, and the melting points have not always been definitive (1). Chromatographic methods have aided in establishing the homogeneity of porphyrins and porphyrin esters (2-5). Nevertheless, the multiple porphyrins which may result from the conditions required to convert hemins to porphyrins (1,6,7) and the instability of some porphyrins make this general procedure for hemin identification less certain than if the hemins themselves could be identified. More recently, methods of column chromatography have been developed which can separate protohemin from hemin a (8) and even from two hemin a types (9). These techniques were essentially preparative, but a microprocedure for the identification of hemin prosthetic groups prepared from small amounts of hemoproteins would seem useful. Chu and Chu (10) have reported solvent systems for the separation of hemins by paper chromatography, but the hemins used were pure samples which had been prepared by large scale purification procedures. The method reported here deals with hemins obtained directly from small quantities of cytochrome c (II), cytochrome oxidase preparations (12)) hemoglobin, catalase (13)) and various rat tissues. Special procedures for the separation of the hemins from their hemoproteins are reported, and new acid solvent systems for the paper chromatography of the hemins are described. The separation of various hemins from the above preparations and tissues is demonstrated.