At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5' donor splice junction of intron 14 and the 3' end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOHterminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.

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A collaborative search for albumin genetic variants (alloalbumins) was undertaken by cellulose acetate and agarose electrophoresis at pH 8.6 of the sera of patients at two major medical centers in the United States and of nearly 20,000 blood donors in Japan. Seventeen instances of alloalbuminemia were ascertained, and seven different alloalbumin types were characterized by structural study. Two previously unreported alloalbumin ypes were identified. In one (8)(9)(10)(11)(12)(13)(14)(15). Ofthe nearly 4000 possible effective point mutations in the 585 codons of the albumin gene, about 800 would lead to a change in electrophoretic mobility. Yet, only about 35 different alloalbumins have been detected and characterized structurally. In continuation of our investigation of albumin as a model for study of neutral protein evolution (5-7, 9, 16-21), we report collaborative research on alloalbumins identified in two population groups: (i) by clinical electrophoresis at two major medical centers in the United States and (ii) by screening for alloalbumins in the sera of some 20,000 blood donors in Japan.Seventeen instances of alloalbuminemia in unrelated individuals were detected, and seven structurally different alloalbumins were identified and characterized in this investigation. Two previously unreported alloalbumins were identified. One of these designated Iowa City-1, which was present in a Caucasian family, had the substitution 365 Asp Unexpectedly,-a Naskapi-type alloalbumin (372 Lys Glu) was found in a single Japanese; this variant albumin had previously been reported only in Athabascan-and Algonkianspeaking Amerindian tribes in which it occurs with a polymorphic allele frequency-i.e., a frequency -1% and in Eti Turks (3, 17). MATERIALS AND METHODSElectrophoretic Screening. Detection of albumin variants in the three surveys was done by cellulose acetate and agarose electrophoresis at pH 8.6. This procedure was also used for comparison to reference alloalbumins with known substitutions and after limited tryptic digestion to identify possible proalbumins (16). The 11 bisalbuminemic sera from the Mayo Clinic were accumulated over a period of 17 years and were noted during routine clinical electrophoresis because of the slow (+2 mobility units) or fast (-2) mobility of the variant albumin, and no alloalbumins with a change of + 1 or -1 mobility units are represented. The three alloalbumins in the Iowa City series were identified by inspection of electrophoretograms of sera of patients and staff over a period of about 4 years. The Japanese (Komagome) project consisted of electrophoretic screening for albumin variants in the sera of 18,874 blood donors at the Tokyo Metropolitan Blood Center of the Japanese Red Cross.Structural Studies. Because of the number and variety of the specimens reported here and the fact that most of the methods have been published in detail (5)(6)(7)(16)(17)(18)(19)(20)(21), only a summary of the methods is given for each type of alloalbumin. The general strategy for structural study...

The rhesus monkey, Macaca mulatto, exhibits a geographically restricted polymorphism of serum albumins Mac A and Mac B that is recognized by electrophoresis and is associated with a difference in bilirubin-binding parameters. To identify the basis of the polymorphism, the cDNA and protein sequences of serum albumin from M. mulatta were determined. Screening of a Agtll rhesus liver cDNA library yielded a 1988-bp cDNA sequence that encodes the complete amino acid sequence of mature albumin, the entire propeptide, and part of the prepropeptide. Isoelectric focusing and aminoterminal protein sequencing of CNBr fragments of albumin from A/A and B/B homozygotes were performed, and the structural difference was localized to a CNBr fragment (MCB3) spanning residues 124-264. (5), who determined the crystallographic structure of human serum albumin at a resolution of 2.8 A. One approach to investigating binding sites is to study structural differences in genetic variants that differ in specificity of binding for a particular ligand. In this paper we report the amino acid and cDNA sequence of the serum albumin of the rhesus monkey, Macaca mulatta,l1 and the difference in sequence of two polymorphic forms of macaque albumin that differ in bilirubin binding specificity (6-10).It is a paradox that although analbuminemia is extremely rare but apparently tolerable (1, 11), genetic variants of albumin (alloalbumins) are also extremely rare in humans and other species (11-17). More than 40 different human alloalbumins have been identified by protein or DNA sequence analysis, many of them in our laboratory (11-17); however, their cumulative frequency worldwide is only of the order of 1 in 3000. Occasional reports of albumin polymorphism in domestic animals that are based on electrophoretic analysis have appeared, but none of these has apparently been substantiated by structural study. In contrast, by electrophoretic analysis Smith and co-workers (6-9) have identified a pronounced geographic distribution of albumin polymorphism in wild populations of macaque monkeys. Segregation analysis ofthe two phenotypes (Mac A and Mac B) in M. mulatta from India is consistent with the hypothesis of two codominant alleles (Al.ac and Allac) whose frequencies are approximately 0. 3-0.4 and 0.6-0.7, respectively (6, 10

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