The protein HC chromophore is linked to the cysteine residue at position 34 of the polypeptide chain by a reduction-resistant bond and causes the charge heterogeneity of protein HC.
Protein HC, an extremely charge-heterogeneous lipocalin, carries a yellow-brown fluorescent chromophore of unknown structure covalently bound at an unidentified site of its polypeptide chain. Two chromophore-carrying peptides with 60% of the chromophore material (defined as material absorbing light at 330 nm) were isolated from pepsin-digested native human protein HC by reversed-phase high performance liquid chromatography. Sequence analysis of these peptides indicated that the chromophore was bound to the cysteine residue at position 34 of the protein HC polypeptide chain. Sequence analysis of a native chromophore-tripeptide complex, isolated from pronase digests of the pepsin-produced peptides, identified the sequence Thr33-X34-Pro35, corroborating the position of the chromophore linkage. Quantitative amino acid analysis of the hydrolyzed, performic acid-oxidized, chromophore-tripeptide complex demonstrated approximately equal amounts of threonine, cysteic acid, and proline in the complex. Reduction and carboxymethylation of the native chromophore-tripeptide complex did not remove the chromophore from the peptide. The absorption spectrum of the chromophore-tripeptide complex was similar to that of native protein HC, implying that all of the heterogeneity of protein HC resides in its chromophore.
Escribano
J
J
Servicio de Endocrinologia, Centro Ramón y Cajal, Madrid, Spain.
Grubb
A
A
Calero
M
M
Méndez
E
E
D000478:Alkylation; D000510:Alpha-Globulins / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D000510:Alpha-Globulins; C016001:alpha-1-microglobulin; D010434:Pepsin A; D003545:Cysteine