PURIFICATION AND PROPERTIES OF SALMON LIVER BILIVERDIN REDUCTASE

Abstract

Biliverdin reductase (BVR: bilirubin:NAD(P)+ oxidoreductase; EC 1.3.1.24) is responsible for the conversion of biliverdin to bilirubin a t the end of the haem catabolic pathway. Elevated levels of serum bilirubin (hyperbilirubinaemia) occur when bilirubin conjugation is impaired. When the bilirubin carrying capacity of the blood (mediated by serum albumin) is exceeded, the pigment becomes deposited in fatty tissues such as those of the CNS where the pigment is neurotoxic, leading to kernicterus. While the enzyme is present in mammals and some species of fish, it is apparently absent from birds, reptiles and amphibians. There has been some question as to the benefit of this pathway as the pigment is considered to be a toxic metabolite in phyla that produce it while other higher vertebrates apparently function successfully in its absence. However, there is increasing evidence that bilirubin may play a significant role a s an antioxidant Ill. The purification of BVR from a number of mammalian sources (ox 121, rat 131, pig (41 and human 151) has been reported and others have reported BVR activity in various species of fish 161. We have been interested in the phylogenetic development of this protein and its apparent absence in birds and reptiles and have therefore embarked on a study of the salmon BVR to determine it's relatedness to the mammalian enzyme. One kg of salmon liver was homogenised in buffer A (20mM Tris/HCI. pH 7.3: 150mM NaC1: ImM EDTA) and after centrifugation a t 13,600 g for 30 minutes the supernatant was subjected to ammonium sulphate fractionation (40-70%). This material was dialysed against three changes of buffer B (10 mM sodium phosphate pH 7.2) and then loaded onto a column of DEAE-cellulose (16 x 7 cm) equilibrated in the same buffer. The enzyme was eluted using a gradient of 0 to 120 mM KC1 (2 by 1.5L). Active fractions were then precipitated with 70% ammonium sulphate and dialysed against buffer C (10mM sodium phospate pH 7.2; lOOmM KCl). After centrifugation at 20,000 g for 10 minutes the supernatant was loaded onto a Sephaclyl S-200 column (120 x 4.5 cm) and eluted with the same buffer. This material was diluted 1:1 with lOmM sodium phosphate buffer, pH 7.2 and then applied to a column of 2',5'ADP-Sepharose. After washing with the equilibration buffer (lOmM sodium phosphate pH 7.2: 50mM KCl) the enzyme was eluted using a 0.05-1.OM KCl gradient (2 by 200ml). This material was m e d for kinetic studies; to raise monospecific polyclonal anti-serum in NZW rabbits: and to generate peptides for amino acid sequencing. Fig. 1 shows the results of DEAE-cellulose chromatography. The enzyme reproducibly elutes a s two peaks, with a possible third peak forming a shoulder late in the second peak (Fig. 1). All further purification work was carried out using the second major peak (P2). BVR eluted as a single peak on S-200 gel filtration. corresponding to a relative molecular mass of 34.000. After 2'5' ADP-Sepharose chromatography, the enzyme was homogeneous on SDS-PAGE with a M, of 34.000 (Fig. 2). Preliminary sequence determination on cyanogen bromide fragments has presented evidence that the salmon enzyme shares significant relatedness to the rat enzyme (although this is not reflected in immunological activity). In Fig. 3 the rat sequence 17) is shown and fragments that have been tentatively identified as revealing partial identity are marked. Salmon BVR appears to be structurally related to the rat enzyme which raises some interesting phylogenetic questions as the enzyme is not reported to exist in amphibians, birds or reptiles. Further work is necessary to confirm the latter claim, however it raises the possibility that the BVR gene may exist albeit in a non-functional form in these species. Alternatively BVR may be an example of convergent evolution a t a molecular level.