The chemiluminescence from the oxidation of the thiol in albumin

Abstract

Blood serum emits a low intensity chemiluminescence (10 - 10 4 hv cm-2 s-1), the majority of which is produced from the plasma protein albumin. It has been suggested that albumin emits light through reactions that involve the oxidation of its free thiol, the cysteine - 34 residue, with the subsequent production of hydrogen peroxide. This study used a highly sensitive photomultiplier detector to measure the chemiluminescence intensities of a variety of albumins under different conditions, in order to test this hypothesis. A direct relationship was observed between the thiol content and the chemiluminescence from albumins. The intensity of the chemiluminescence from a defatted bovine serum albumin sample was ca. two thirds that from a native bovine albumin sample, the same ratio as the difference in thiol contents from native and defatted albumins. The positioning of the thiol in a less sterically hindered position was correlated with an increase in chemiluminescence. The reactions of ligands that bind to the thiol changed the intensity of the chemiluminescence, showing the involvement of the thiol in producing chemiluminescence. The magnitude of the chemiluminescence quenching from fatty acids depended on the fatty acid chain length and therefore likely binding site. Fatty acids alter the conformation of the albumin molecule, affecting the ease of oxidation of the thiol. The intensity of the chemiluminescence from different albumins was correlated to the specificity of the copper binding site, with albumins for which the binding site has a high affinity for Cu(II), producing a less intense light emission. The Cu(II) cation may catalyse oxidation of the thiol. The metal chelator EDTA had a maximum effect of ca. 50% on albumin regardless of the Cu(II) binding geometry, suggesting that the chemiluminescence may proceed at up to half the normal rate in the absence of Cu(II). Results from this work support the hypothesis that the thiol plays a major role in the chemiluminescent reactions of albumin, and show that the chemiluminescence is affected by fatty acids that bind to the albumin molecule, and by the specificity of the Cu(II) binding site in the N-terminal cleft that contains the free thiol.