The pigment contained in the extracts obtained from B. phosphorescens by freezing and thawing, and in the alkaline extracts of B. phosphorescens and yeast, resembles the "cytochrome c" of Hill and Keilin (6) and the "porphyratin B" of Schumm (7) in giving absorption bands at mµ 552-550 and 522-520) but shows in addition a band about 575, as in the "hemochromogen A" obtained by Keilin (3) by prolonged treatment of yeast with strong alkali. Like cytochrome c the pigment of yeast extracts appears to be distinct from the ordinary hemochromogen of blood, because of the difference in position of the bands of the native materials and of the corresponding pyridine hemochromogens. On treatment with acetic acid, however, the yeast extract yields α-hematin, as identified spectroscopically. It is evident then that one portion of its iron-porphyrin nucleus is identical with α-hematin (iron-protoporphyrin), which must be present not as such, but in chemical combination.
The alkaline extracts of C. diphtheriae, compared with those of B. phosphorescens and yeast, show a constant difference in the position of the two bands in the green, which lie nearer the red end of the spectrum, at mµ 556 and 528. This extract likewise on treatment with acetic acid yields α-hematin, which in the form of its alkaline hemochromogen may be responsible for the bands in the alkaline extract at mµ 556 and 528.
Great interest has attached in our investigation to the substance responsible for the absorption band in the alkaline extracts about 575. Extraction with acetic acid-ether of these alkaline solutions, as well as of the whole bacteria, yields a material which shows absorption bands at mµ 575-574 and 539-535, and appears to be identical with a complex porphyrin which has been found in culture filtrates of C. diphtheriae.
This complex porphyrin has been described in a previous paper (1). It is labile and breaks down readily to yield coproporphyrin and the copper compound of coproporphyrin, and is apparently the source of the coproporphyrin which is often found free in the culture filtrates. In the work repeated earlier we had been unable to obtain this complex porphyrin, or porphyrin compound, directly from the bacteria. In the present work we have been successful in obtaining it from the three species investigated.
The behavior of the complex porphyrin extracted from the whole bacteria is the same as of that found in filtrates. It is insoluble in 25 per cent HCl, and on disintegration gives coproporphyrin and the copper compound of coproporphyrin.
Information is quite lacking as to the particular form of combination in which this complex porphyrin occurs within the cell. The complex porphyrin is certainly not present there in the form in which it appears in the extracts. If diphtheria bacilli showing strong absorption bands of reduced cytochrome, while under examination with the microspectroscope are treated with glacial acetic acid, the bands of cytochrome are seen to fade and are replaced by those of the complex porphyrin at mµ 575 and 539.
The origin of the copper which is found, combined with coproporphyrin, as a product of the disintegration of the porphyrin compound, has been a matter of uncertainty. In the case of filtrates of C. diphtheriae it has seemed possible that the copper was never a constituent of the bacteria, and that combination with copper occurs only after the porphyrin has been liberated from the bacterial cell. With washed bacteria, however, the presence of copper in extracts indicates that this element has been taken up from the culture medium and incorporated within the cell. Whether or not the copper is there combined with porphyrin cannot be decided by the present evidence. Copper occurs naturally, however, in combination with porphyrin in turacin (14), a pigment of the wing feathers of certain birds. In the present case such combination seems the more probable, so that the complex porphyrin may represent a form in which copper is contained within the cell.
Objection may be raised to the use of the term complex porphyrin or porphyrin compound for the substance referred to here and in the previous paper (1). The name hemochromogen might be applied with equal justification. Until the chemical nature of the substance is better known, however, it seems best not to use any but a simple descriptive name.
Reference should not be omitted here to the bacteriological significance of this compound, which arises from the correlation which we have previously observed between its amount and the content of toxin, in filtrates of C. diphtheriae. In respect to this porphyrin compound the pathogen C. diphtheriae seems to differ from the nonpathogenic forms in the readiness with which the material is liberated from the bacteria in cultures, rather than in the nature of the material.