alpha(1)-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound

alpha(1)-Microglobulin (alpha(1)m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of protein s with a three-dimensional (3D) structure that forms an internal hydrophobi c ligand-binding pocket. alpha(1)m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extrem ely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alp ha(1)m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified ch romophores with molecular masses between 122 and 282 atomic mass units (amu ). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also puri fied. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modifi ed. The results can explain the size, charge, and optical heterogeneity of alpha(1)m. A 3D model of aim, based on the structure of rat epididymal reti noic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supporte d by the fluorescence spectra of alpha(1)m under native and denatured condi tions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha(1) m is complex bound to IgA. Only the free alpha(1)m carried colored groups, whereas alpha(1)m linked to IgA was uncolored.