Photooxidation of Human Serum Albumin and Its Complex with Bilirubin

Abstract

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole 0 2 , and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen.Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole 0 2 . Parallel photooxidation of the protein carrier could not be detected.It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.

Antioxidant activity of albumin-bound bilirubin.

“…1C) and the calculated rate of radical productions indicates that, initially, all radicals formed are scavenged by bilirubin, thereby completely protecting albumin-bound fatty acids and, most likely, the protein itself from oxidation. The latter notion is supported indirectly by the finding that photooxidation of Alb-BR resulted in substantial oxidation of bilirubin, whereas no oxidation of the protein was observed as judged by amino acid analysis (23).…”

Porphyrin-Induced Photooxidation of Conjugated Bilirubin

“…The affinity constant of fluorescent binding site for bilirubin is 7x10 6 M -1 and one hundred times stronger than the other non-fluorescent binding site. HSA is photooxidized in the presence of the sensitizers, rose bengal or methylene blue, whereas no photooxidation is observed in the presence of bilirubin [20]. In this report bilirubin bound to HSA undergoes phototransition from Z form to E form of bilirubin IXα, which is unable to form internal hydrogen bond and is water-soluble.…”

“…Large amounts of porphyrins and abnormal concentrations of copper are found in neonatal hyperbilirubinaemia patient serum [18,19]. Irradiation of bilirubin in vitro elicits the oxidation of bilirubin [9] and the photooxidation of bilirubin is markedly increased in the presence of photosensitizers [12,14,20]. There is evidence that these oxidations involve singlet oxygen generated through the energy transfer from excited sensitizers to oxygen [10][11][12].…”

Bilirubin-albumin binding affinity and serum albumin concentration during intensive phototherapy (blue double light) in jaundiced newborn infants

“…By in vitro irradiation of human serum albumin in the presence ofa photosensitizer (methylene blue or rose bengal), oxidation of the albumin occurred causing a reduced bilirubin binding affinity [13,15]. In the presence of bilirffbin, which is another photosensitizer, the same occurred during intensive irradiation [16].…”