242 VI. HEMOGLOBIN 



phenomena. While caffeine, 1-methylimidazole, or pilocarpine cause sharpen- 

 ing of the absorption bands in the visible region of the spectrum, bile salts, 

 digitonin, or sodium dodecyl sulfate are without effect. It is improbable 

 that the presence of the copper atom or the side chain differences between 

 uro- and protoporphyrin would affect the ability of the flat porphyrin plate 

 to form van der Waals' compounds with the above-named substances. The 

 copper atom would, however, prevent linkage of these substances to the 

 pyrrole nitrogens. 



While the carboxylic acid side chains may be involved in the formation 

 of compounds of protoporphyrin and turacin with caffeine, there is evidence 

 that in the formation of the caffeine heme compounds {150If) the iron atom is 

 also involved. The caffeine heme compound is not a hemochrome, the 

 spectral change on combination being similar to that observed when caffeine 

 is added to porphyrin. Caffeine combines with heme and with carbon 

 monoxide heme, but does not combine with hematin. The affinity of caffeine 

 for heme is increased when the carbon monoxide compound is formed. If 

 excess caffeine is added to pyridine hemochrome, the spectrum slowly changes 

 to that of the caffeine heme compound, while addition of more pyridine 

 re-forms the hemochrome. 



It is difficult to understand this competition if the caffeine and the pyridine 

 are not both attached to the iron atom. We do not believe that this possibility 

 invalidates our earlier conclusions from the caffeine protoporphyrin and the 

 caffeine turacin cpmpounds that caffeine combines with the carboxyl side 

 chains. In the caffeine heme compound the caffeine may be combined to 

 both the iron and the carboxyl side chain. Keilin tested a number of purines, 

 pyrimidines, imidazole derivatives, and several alkaloids. Besides caffeine 

 only chlorocaffeine showed a simple "caffeine effect," while pilocarpine which 

 is able to produce this effect, is also able to form a hemochrome. 



As with the compounds of globin with the porphyrins and nonferrous 

 metalloporphyrins, the combination of caffeine with mesoheme, hematoheme, 

 and deuteroheme excludes the vinyl side chains from any active role. 



Most of the evidence for combination with carboxyhc acid side 

 chains in this and in the previous sections is based on spectroscopic 

 observations. The evidence is also indirect in that, with the exception 

 of the caffeine heme compound, the carboxyhc acid group is left as 

 the most probable point of attachment, after hnkage to other parts 

 of the molecule has been excluded by working with different deriva- 

 tives. The crucial experiments with porphyrins or metalloporphyrins 

 lacking carboxyl groups have not yet been reported, probably owing 

 to the difficulty of working with these substances in aqueous solution. 

 On the other hand there is no evidence that in addition to the essential 

 iron imidazole linkage in the hemoglobin, secondary carboxyl protein 

 linkages may not be formed. The sharpening of the absorption bands, 

 in consequence of the breaking up of polymers, by combination of 



