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 variant 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). This fragment was isolated on a preparative scale and subjected to tryptic and V8 protease digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution Lys240-->Glu. The -2 charge change of albumin Herborn, which is probably due to a A-->G transition in the first position of the corresponding codon in the structural gene, has no significant effect on its electrophoretic mobility under non-denaturating conditions. Therefore we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 453-459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The apparent equilibrium association constants (Ka +/- SD) and the number of high-affinity binding sites (n) of the defatted variant for bilirubin and biliverdin were Ka = 1.03 +/- 0.18 x 10(8) M-1, n = 1.07; and Ka = 7.48 +/- 1.10 x 10(6) M-1, n = 1.01, respectively. The Ka values are about 93.3% and 99.1% of the values found for the normal protein under the same conditions. These results strongly suggest that Lys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high-affinity site.
Dipartimento di Biochimica, Università di Pavia, Italy.
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Minchiotti
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Galliano
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Zapponi
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Tenni
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001663:Bilirubin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D005858:Germany; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D000075462:Serum Albumin, Human; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D012709:Serum Albumin; C081760:albumin Herborn; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003488:Cyanogen Bromide; D001663:Bilirubin; D000075462:Serum Albumin, Human