Comp. Bimhem Physiol., Vol. 62B, pp. 305 to 308. 0305-0491 79 0401-0305502.00/0 © Pergamon Press Ltd 1979. Printed in Great Britain HEMOGLOBIN POLYMORPHISM IN EQUUS PRZEWALSKII AND E. CABALLUS ANALYZED BY ISOELECTRIC FOCUSING OLIVER A. RYDER Zoological Society of San Diego, Research Department, PO Box 551, San Diego. California 92112 U.S.A.: ROBERT S. SPARKES and MARYELLEN C. SPARKES School of Medicine, Department of Medicine, University of California, Los Angeles, California 90024 U.S.A. and JOHN B. CLEGG Oxford University, Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford OX2 6HE, U.K. (Receired 30 August 19781 Abstract 1. Through the use of isoelectric focusing and peptide analysis, the hemoglobins of Prze- walski's horse, Equus przewalskii and the domestic horse, E. caballus have been compared. 2. Przewalski's horses have two separate ~-globin chain polymorphisms similar to domestic horses. Each hemoglobin phenotype could be accurately determined by isoelectric focusing. 3. Confirmation of the electrofocusing hemoglobin determinations was made by comparison to amino acid composition analyses of purified tryptic peptides and by analysis of the rare hemoglobins pheno- types observed in a family of Norwegian trotting horses. 4. Hemoglobin genotypes of fifteen Przewalski's horses were determined and inheritance of hemo- globin haplotypes has been observed. INTRODUCTION Przewalski's horse, Equus przewalskii is the only true wild horse surviving to the present day. Restricted to the western Takin-Shara-Nuru range in the Mon- golian Peoples Republic in recent times, its former range extended throughout Europe and Asia (Mohr, 1970). Some 270 of this endangered species survive in captivity (Volf, 1976) in zoos and animal parks. Comparative analyses of E. przewalskii and the domestic horse, E. caballus, utilizing a variety of mor- phological and biochemical criteria have established the close similarity of these two species (Simpson, 1950: Trommershausen-Smith et al., 1979). Cytogene- tic studies of domestic and Przewalski's horses have shown that the karyotypes of these two species differ by a Robertsonian translocation. Comparative chromosome banding studies of these two species have revealed that fusion of four acrocentric chromo- somes of E. przewalskii forms two metacentric chromosomes (pair 5) in E. caballus (Short et al., 1974; Ryder et al., 1978). Domestic horse hemoglobins have been shown by Kilmartin & Clegg (1967) to exhibit two distinct ~t-chain polymorphisms. Substitution of lysine (lys) or gutamine (glnl at position 60 of the ct-globin chains (ct60) produce, respectively, the slow or fast hemo- globins detectable by starch gel electrophoresis (Kil- martin & Clegg, 1967). Normally, the fast form accounts for 60°4, of the total hemoglobin, and the slow form 40~o. Additionally, analysis of amino acid compositions of tryptic peptides of purified ~-globin chains revealed a polymorphism at position 24 (ct24) in which both phenylalanine (phe) and tyrosine (tyr) residues may occur. Further genetic and biochemical studies by Clegg (1970) have shown that four distinct, heritable, a-chain sequences exist, depending on the substitutions at positions 24 and 60. All /3-chain sequences examined were apparently identical. Isoelectric focusing of hemoglobins has proved to be a powerful tool in separating sequence variants in humans (Drysdale et al., 1971: Jeppson et al., 1972: Monte et al., 1976) and other species as well (Butcher & Hawkey, 1977). We have utilized isoelectric focus- ing techniques to compare the hemoglobins of Prze- walskFs and domestic horses and investigate whether similar polymorphisms occur in these two species. These studies have revealed that the four ~-chain sequences present in E. caballus are also present in E. przewalskii and, furthermore, each phenotype can be accurately determined by isoelectric focusing with- out resorting to amino acid composition analysis of separated tryptic peptides. Electrofocusing analysis was also able to accurately identify the rare hemo- globin variants found in Norwegian trotting horses by Braend (19671 and further characterized by Clegg (1970). MATERIALS AND METHODS Blood samples from E. caballus were obtained from nor- mal adult animals by venipuncture. Blood samples from E. przewalskii were obtained by venipuncture of adult ani- mals which had been chemically immobilized for routine veterinary procedures. Isoelectric focusing was performed in thin polyacryl- amide slab gels on a commercial apparatus (LKB Multi- 305