BILE PIGMENT CHROMOPROTEINS 147 



An excellent starting material is the Japanese delicacy "nori," which 

 consists of carefully purified and dried Porphyra tenera Kjelm. (Bangiaceae) 

 and was first used by Kitasato (154'£)- Similar algae are said to be harvested 

 and used as a foodstuff under the name "laver" in England or "stoke" in 

 Ireland {2Jt30). Ceramium rubrum (Florideae) and the fresh water alga 

 (Aphanizomenon flos aquae) were also used. 



Kylin and Kitasato made unsuccessful attempts to obtain clues to the 

 nature of the prosthetic pigment group. They hydrolyzed the chromopro- 

 teins with proteolytic enzymes and with acid and alkali. The compounds 

 obtained by their methods were, however, not the free prosthetic groups 

 but still rather large polypeptides. This explains the failure of Kitasato to 

 obtain pyrrole bases on hydriodic acid reduction of this "prosthetic group" 

 of phycoerythrin and also later erroneous results of Levene and Schormiiller 

 {1723, cf. 1690). 



The prosthetic group is bound much more firmly than is heme in 

 hemoglobin and can only be set free by drastic action of acid {1671, 

 1673), or by treatment with alkali which, however, causes secondary 

 alteration {1689,1690). The nature of the prosthetic groups, phy- 

 cocyanobilin and phycoerythrobilin, as bile pigments and their iden- 

 tity with mesobiliviolin and mesobilierythrin was established by 

 Lemberg {1671,1673,1689,1690); their chemical structure has already 

 been discussed in Sections 5.2. and 5.3. The linkage between pros- 

 thetic group and protein appears to be a peptide linkage between the 

 propionic acid side chains of the prosthetic groups and amino groups 

 of the protein. 



There isj however, evidence for a second labile linkage, broken by 

 mild acid treatment. The native chromoproteins, which are metal- 

 free, do not combine with zinc although the free prosthetic groups 

 and also the chromoproteins denatured by mild acid treatment do so. 

 The pyrromethene grouping which reacts with the metal is masked 

 in the intact chromoprotein, but is set free very early. At the same 

 time the strong fluorescence of the native .chromoproteins disappears. 



From the yield of phycocyanobilin (mesobiliviolin), Lemberg {1672) con- 

 cluded that a molecule of C-phycocyanin contained eight molecules of the 

 prosthetic group. According to this the molar extinction coeflBcient of phy- 

 cocyanin (per mole of prosthetic group) would be extraordinarily high 

 (*mM~ 332). A large decrease of the extinction on liberation of the pros- 

 thetic group, and even subsequent to treatment of the chromoprotein with 

 dilute mineral acid was indeed observed (Lemberg, 1673), but nevertheless 

 it appears more likely from a comparison of the extinction coeflScients of 

 mesobiliviolin with that of phycocyanin that one molecule of the chromo- 

 protein contains sixteen, not eight, molecules of mesobiliviolin. 



Table XI gives a summary of individual phycoerythrins and phyco- 



