THE STRUCTURAL CHARACTERIZATION AND BILIRUBIN-BINDING PROPERTIES OF ALBUMIN HERBORN, A [LYS240-]GLU] ALBUMIN MUTANT

We report the molecular defect of albumin Herborn, a new genetic varia nt of human serum albumin which has been found in Germany. Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr 3 (residues 124-298). Th is fragment was isolated on a preparative scale and subjected to trypt ic and V8 protease digestion. Sequence determination of the abnormal t ryptic and V8 peptides revealed that the variant arises from the subst itution Lys240-->Glu. The -2 charge change of albumin Herborn, which i s probably due to a A-->G transition in the first position of the corr esponding codon in the structural gene, has no significant effect on i ts electrophoretic mobility under non-denaturating conditions. Therefo re we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 4 53-459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The a pparent equilibrium association constants (K(a) +/- SD) and the number of high-affinity binding sites (n) of the defatted variant for biliru bin and biliverdin were K(a) = 1.03 +/- 0.18 x 10(8) M-1, n = 1.07; an d K(a) = 7.48 +/- 1.10 X 10(6) M-1, n = 1.01, respectively. The K(a) v alues are about 93.3% and 99.1 % of the values found for the normal pr otein under the same conditions. These results strongly suggest that L ys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high-af finity site.