Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin.
C-Phycoerythrin is a light-harvesting protein whose alpha and beta subunits carry thioether-linked phycoerythrobilin (PEB) at cysteine residues alpha-82, alpha-139, beta-48,59 (doubly-linked), beta-80, and beta-165. The two subunits of Calothrix sp. PCC 7601 C-phycoerythrin, overexpressed together as apopolypeptides in Escherichia coli, formed inclusion bodies. Purified apo-alpha was soluble in the absence of urea, whereas the apo-beta subunit was only soluble at high urea concentrations. Products of nonenzymatic addition of PEB and phycocyanobilin (PCB) to apo-alpha were characterized by isolation of bilin peptides and spectroscopy. Reaction of PEB with the apo-alpha subunit led primarily to 15,16-dihydrobiliverdin (Cys-82) or urobilin (Cys-139) adducts, and small amounts of the natural PEB adducts at both Cys-82 and Cys-139. PCB reacted primarily with Cys-82 to form phycocyanobilin and mesobiliverdin adducts. Both PEB and PCB also formed relatively small amounts of adducts with Cys-59, which is not a bilin attachment residue in natural phycoerythrin. Sodium azide was found to promote the addition of PEB to simple thiols but not to apo-alpha phycoerythrin.
Fairchild
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Glazer
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D001386:Azides; D001654:Bile Pigments / Q000737:chemistry*; D004798:Enzymes / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D010799:Phycoerythrin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019810:Sodium Azide; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds; D014357:Trypsin; D014508:Urea
D001386:Azides; D001654:Bile Pigments; D004798:Enzymes; D013438:Sulfhydryl Compounds; D010799:Phycoerythrin; D014508:Urea; D019810:Sodium Azide; D014357:Trypsin