Structural characterization, stability and fatty acid-binding properties of two French genetic variants of human serum albumin Lorenzo Minchiotti a , Ulrich Kragh-Hansen b , Henning Nielsen b , Elisabeth Hardy c , Anne-Yvonne Mercier c , Monica Galliano a ; * a Dipartimento di Biochimica `A. Castellani', University of Pavia, I-27100 Pavia, Italy b Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark c Etablissement de Transfusion Sanguine de Bretagne Occidentale, F-29275 Brest Cedex, France Received 23 October 1998; received in revised form 14 January 1999; accepted 28 January 1999 Abstract Four bisalbuminemic, unrelated persons were found in Bretagne, France, and their variant and normal albumins were isolated by DEAE ion exchange chromatography, reduced, carboxymethylated and treated with CNBr. Comparative two-dimensional electrophoresis of the CNBr digests showed that three of the variants were modified in fragment CB4, whereas the fourth had an abnormal fragment CB1. These fragments were isolated, digested with trypsin and mapped by reverse-phase HPLC. Sequencing of altered tryptic peptides showed that the three variants modified in CB4 were caused by the same, previously unreported, amino acid substitution: Asp 314 CVal (albumin Brest). The fourth, however, was a proalbumin variant with the change Arg 32 CCys (albumin Ildut). Both amino acid substitutions can be explained by point mutations in the structural gene: G ATCG TT (albumin Brest) and CGTC TGT (albumin Ildut). The proalbumin Ildut is very unstable and already in vivo it is to a large extent cleaved posttranslationally to Arg-Albumin and normal albumin. Furthermore, we observed that during a lengthy isolation procedure the remaining proalbumin was changed to Arg-Albumin or proalbumin lacking Arg 36 . In addition, part of normal albumin had lost Asp 1 . Gas chromatographic investigations using isolated proteins indicated that albumin Brest has improved in vivo fatty acid-binding properties, whereas the structural modification(s) of albumin Ildut does not affect fatty acid binding. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Human serum albumin; Proalbumin; Genetic variant; Peptide mapping; Single amino acid substitution; Fatty acid binding 1. Introduction Human serum albumin is a single-chain, unglyco- sylated protein of 585 amino acids, which is synthe- sized in the liver as preproalbumin. The 18-residue prepeptide is cleaved o¡ by a signal peptidase as the polypeptide chain enters the lumen of the endoplas- matic reticulum [1]. When the resulting proalbumin reaches the Golgi vesicles, the Arg-Gly-Val-Phe-Arg- Arg propeptide is hydrolyzed by a membrane-bound proalbumin convertase to form the mature protein with aspartic acid as the amino-terminal residue [2]. Normally, proalbumin is not detectable in the blood- 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00026-6 Abbreviations : Alb A, normal (wild-type) human serum albu- min; C m:n , fatty acid composed of m C-atoms and having n dou- ble bonds; NEFA, non-esteri¢ed fatty acids; pI, isoelectric point; RP, reverse phase * Corresponding author. Fax: +39 (382) 423108; E-mail : galliano@unipv.it Biochimica et Biophysica Acta 1431 (1999) 223^231