Comp. Bimhem Physiol., Vol. 62B, pp. 305 to 308. © Pergamon Press Ltd 1979. Printed in Great Britain
0305-0491 79 0401-0305502.00/0
H E M O G L O B I N P O L Y M O R P H I S M IN EQUUS PRZEWALSKII A N D E. CABALLUS A N A L Y Z E D BY ISOELECTRIC F O C U S I N G OLIVER A.
RYDER
Zoological Society of San Diego, Research Department, PO Box 551, San Diego. California 92112 U.S.A.: ROBERT S. SPARKES a n d 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 Przewalski'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 phenotypes observed in a family of Norwegian trotting horses. 4. Hemoglobin genotypes of fifteen Przewalski's horses were determined and inheritance of hemoglobin 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 Mongolian 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 morphological and biochemical criteria have established the close similarity of these two species (Simpson, 1950: Trommershausen-Smith et al., 1979). Cytogenetic 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 chromosomes 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 hemoglobins detectable by starch gel electrophoresis (Kilmartin & 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 focusing techniques to compare the hemoglobins of PrzewalskFs 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 without resorting to amino acid composition analysis of separated tryptic peptides. Electrofocusing analysis was also able to accurately identify the rare hemoglobin variants found in Norwegian trotting horses by Braend (19671 and further characterized by Clegg (1970).
305
MATERIALS AND M E T H O D S
Blood samples from E. caballus were obtained from normal adult animals by venipuncture. Blood samples from E. przewalskii were obtained by venipuncture of adult animals which had been chemically immobilized for routine veterinary procedures. Isoelectric focusing was performed in thin polyacrylamide slab gels on a commercial apparatus (LKB Multi-
OLIVER A. RYDER et al.
306
I
2
3
4
5
6
7
8
9
I0
Fig. 1. Isoelectric focusing of hemoglobins from E. przewalskii and E. caballus. Packed red cell hemolysates were subjected to isoelectric focusing as described in Materials and Methods. Slot 1: human HbA: slot 2: E. caballus--Clydesdale (BI/BI): slot 3: E. caballus--Shetland pony (BI/BI): slot 4: E. przewalskii "Bogatka" (BI/BII); slot 5: E. przewalskii "Henrietta" (BII/BII); slot 6: przewalskii "Jeanhold" (BII/BII); slot 7: E. przewalskii "Borkas" (BI/BII); slot 8: E. przewalskii "Bolinda" (BI/BI); slot 9: E. przewalskii "Roland" (BI/BII); slot 10: E. caballus--American standard breed (BI/BII). Breed designations are noted for E. caballus individuals; house names are given for E. przewalskii individuals. Hemoglobin genotypes (Clegg, 1970) as determined by electrofocusing are given in parentheses.
phor 2177). The gel was prepared according to the procedure recommended by the manufacturer (LKB Application Note 75, March 29, 1973) from the following reagents: 7.5g sucrose in 36.6ml H20, 10ml 29.17/o acrylamide (Eastman), 10ml 0.9°4, N,N'-methylene bisacrylamide (Eastman), 0.05ml N,N,N',N'-tetramethylethylene diamine (Eastman), 3.0ml pH 6-8 ampholine (LKB) 40% w/v, and 2.0ml 1% ammonium persulfate (Eastman). Twenty pl of a packed red cell hemolysate was added to 50/~1 of 0.1°/,, aqueous KCN. Consistent with the findings of Bunn & Drysdale (1971), it was observed that treatment of hemolysates with KCN was necessary to prevent extra bands. The KCN-treated hemoglobin solution was then absorbed onto 5 x 8 mm Whatman No. 3 filter paper and placed onto the gel approximately 2 cm from the cathodal Side of the gel. The electrophoresis apparatus was precooled with tap water for at least 30 min before the start of the run. Filter paper strips were saturated with i M H3PO 4 for the anode and 1 M NaOH for the cathode. The gel was run at constant current of 25ma for 30rain. At this time the filter paper which contained the hemolysate was removed. The run was then continued at 900 constant volts for 2 hr or until the current through the gel reached a constant value. Immediately after turning off the power supply, the gel was placed in 12.5~;, trichloroacetic acid for 5min, then rinsed in tap water. The gel was photographed and subsequently stored in water. For chemical analysis red cell lysates were converted to globin by the acid acetone method and the ~-fast,
~t-slow, and fl-chains were fractionated by chromatography on carboxymethyl cellulose in 8 M urea, 0.05 M 2-mercaptoethanol, pH 6.8 as described by Clegg et al. (1966). Separated ~t-fast and or-slow chains were digested with trypsin and the digests fractionated by high voltage electrophoresis on paper at pH 6.5 followed by chromatography in n-butanol: acetic acid: pyridine: water (15: 3: 10:12 by vol) (Kilmartin & Clegg, 1967). Chromatograms were developed by staining with 0.02%(, ninhydrin in acetone and heating at 60°C for 20 min. The ~4 peptides (which include residue 24) to be analyzed were eluted with 6 N HCI containing 2 mg/ml phenol to minimize destruction of tyrosine and hydrolyzed in sealed capillary tubes for 18 hr at 107°C. The hydrolysates were analyzed on an automated analyzer.
RESULTS AND DISCUSSION When KCN-treated packed red cell hemolysates were subjected to electrofocusing, hemoglobin patterns similar to those shown in Fig. l were obtained. Both E. przewalskii and E. caballus showed either two or four major hemoglobin bands. When just two hemoglobin bands were present they are quite well separated. When four hemoglobin bands are present the individual well separated bands are present as doublets (occasionally minor bands also occur even in KCN-treated samples).
Table 1. Correlation of E. przewalskii hemoglobin isoelectric focusing and tryptic peptide compositional analysis
Gel slot in Fig. 1
6 7 8
Species
E. przewalskii E. przewalskii E. przewalskii
Individual
Number of hemoglobin bands observed in electrofocusing
Jeanhold Borkas Bolinda
2 4 2
Amino acid Hemoglobin residue at genotype ~24 (Fig. 3) phe tyr/phe tyr
BII/BII BI/BII BI/BI
Electrofocusing of horse hemoglobins
pH8 $
pH6 ~
,, slow
afast
/ , a e o lys az4
A
tyr
~aso
lys =24 phe
/aso ~aso
gin az4 tyr
gin az4 I~e
307
60
8~
(;If 24 'y'
I
40 af
24 tyr
60
~i
•,/24
I
40 ,",,
tyr
24 tyr
60
/ ' I f 24 ;)he
I
40a,
24 phe
Fig. 2. Interpretation of isoelectric focusing patterns of E. przewalskii and E. caballus.
Fig. 3. Hemoglobin haplotypes in E. cabal/us (Clegg, 1970).
In order to determine whether the four hemoglobin bands detectable by electrofocusing correspond to the four known types of hemoglobin described by Kilmartin & Clegg (1967), analysis of the amino acid composition of purified s-chain tryptic peptides was performed. These results are summarized in Table 1. The Przewalski's horse individuals with two hemoglobin bands exhibited a single residue at position 24 of their ~-chains, having either tyrosine only or phenylalanineonly at this position. There is a clear difference in the isoelectric points reached by the electrofocused fast or slow hemoglobin molecules depending on whether tyrosine or phenylalanine is present (Fig. 1). An individual with four hemoglobin bands was found to have both tyrosine and phenylalanine present at ct24. Furthermore, the isoelectric points reached by four E. przewalskii hemoglobin forms were indistinguishable from those of E. caballus hemoglobins (Fig. 1). Analysis of the remaining tryptic peptide fragments of the or-fast and or-slow chains and from fl-chains in E. przewalskii disclosed no differences in composition from the corresponding peptides obtained from purified E. caballus ct and fl-chains. A diagrammatic interpretation of the electrofocusing and peptide analyses is presented in Fig. 2. The interpretation of the electrofocusing results presented in Fig. 2 was subjected to a further test by electrofocusing hemoglobins from E. caballus individuals with rare variant phenotypes. The unusual hemoglobin phenotypes of these Norwegian horses were first described by Braend (1967) and subsequently by Clegg (1970). They differ from the common pattern in that either the electrophoretically slow hemoglobin molecules are either present in reduced amounts or not at all. Clegg (1970) has suggested that three ~t-chain haplotypes may explain the observed phenotypes. The three postulated haplotypes would each consist of two
linked or-chain genes (Fig. 3). Two of the haplotypes would include genes coding for both lys and gin at ~60, and would code for either phe or tyr at ~t24 of both chains. The proportion of fast and slow hemoglobin bands is 60~o and 40~o respectively. The third haplotypes would contain only gin at ct60 and only tyr at ct24. Thus, if the diagram presented in Fig. 2 is correct, an A/BII pattern should have a 3-banded phenotype with the ~ band being a doublet. Similarly, an A/BI phenotype should be two banded, but the amount of ct~ hemoglobin should be approximately 50~0 of the amount present in a BI/BI homozygote. In Fig. 4, samples of cyanohemoglobin from A/BI and A/BII genotypes have been electrofocused in the same gel with hemoglobins from BI/BI and BII/BII individuals. The data obtained from electrofocusing analysis of the rare hemoglobin phenotypes are consistent with the representation in Fig. 2. Thus, isoelectric focusing of hemoglobins from either E. przewalskii or E. caballus may be used to determine the hemoglobin genotypes without resorting to purification of peptides from globin chains and subsequent amino acid analysis. The patterns obtained upon isoelectric focusing are reproducible and similar patterns may be obtained from fresh samples and samples which have been stored frozen for extending periods. Treatment with KCN before electrofocusing is an important step; incomplete conversion of all hemoglobin molecules present to their cyanohemoglobin forms results in multiple bands due to the differing Oxidation states of the heme iron (Bunn & Drysdale, 1971). The hemoglobin genotypes of fifteen Przewalski's horses from the San Diego Zoo and San Diego Wild Animal Park have been determined by isoelectric focusing (Table II). Inheritance of the hemoglobin haplotypes (Clegg, 1970) in E. przewalskii can be demonstrated through these data. For example, the
I
2
3
4
5
6
7
Fig. 4. Isoelectric focusing of hemoglobins from Norwegian trotting horses with unusual hemoglobin variants. Red cell hemolysates were subjected to isoelectric focusing as described in Materials and Methods. Slot 1: E. przewalskii "Borkas" (BI/BII); slot 2: E. caballus "Lisa" (A/BII); slot 3: E. przewalskii "Bolinda" (BI/BI); slot 4: E. caballus "Torfrid" (30~o ~f, 70~o ct~,phe only at ~t24); slot 5: E. przewalskii "Borkas" (BI/BII); slot 6: E. caballus "Tierra" (A/BI); slot 7: E. przewalskii "Jeanhold" (BII/BII). Hemoglobin genotypes (Clegg, 1970) as determined by direct chemical analysis are given in parenthesis.
308
OLIVER A. RYDER et al.
Table 2. Hemoglobin genotypes of fifteen Przewalski's horses House name Henrietta Bogatka Jeanhold Belkina Belzar Bolinda Bogdo Bellina Borkas Bektari Roxina Belaya Bonnette Roland Hermonia
Sex ~ ~ ,~ ; 3 , ~ ~ : = 5 i
House number WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP CAT ~,
01 02 03 05 06 07 08 10 Il 12 13 14 15 26
Studbook Hemoglobin number genotype 268 504 469 603 638 406 473 319 685 678 480 458 339 320 737
BII/BII BI/BI1 BII/BII BII/BII BII/BII BI/BI BI/BI BI/BII BI/BII BI/BII BI/BI BI/Bll BI/BI BI/BII BI/BII
~'no house number.
individual, Borkas (BI/BII) is the offspring of Jeanhold (BII/BII) and Bolinda (BI/BI) (Table 2). It is, in fact, these three individuals whose hemoglobins are presented together in figure 1. Thus, the hemoglobin haplotypes postulated by Clegg (1970) for domestic horses, E. cahallus, also appear to be inherited in a Mendelian fashion in Przewalski's horse, E. przewalskii. The BI and BII haplotypes appear to be present in these animals at approximately equal frequencies, and the n u m b e r of heterozygotes present does not differ significantly from the proportion expected if the haplotypes are in Hardy-Weinberg equilibrium. Unfortunately, no data on the frequency of BI and BII haplotypes is available for sizable numbers of domestic horses. Therefore, a direct comparison between the relative haplotype frequencies in these two species is presently unknown. With the relative ease that the horse hemoglobin genotypes can be determined by isoelectric focusing, it should now be possible to compile data on haplotype frequency in E. caballus without the effort required for amino acid compositional analysis of tryptic peptides. Additionally, isoelectric focusing should enable hemoglobin genotypes to be used in parentage exclusion determinations in either of the two horse species examined here. As these studies were being conducted, our attention was brought to the report of Kitchen et al. (1976) which showed that the four different E. caballus hemoglobins could be demonstrated by isoelectric focusing.
Acknowledgements--We wish to thank the veterinary staff of the Zoological Society of San Diego, particularly Drs L. S. Nelson, J. E. Oosterhuis, P. T. Robinson and J. E. Meier for their cooperation in obtaining samples.
REFERENCES BRAEND M. (1967) Genetic variation in horse hemoglobin. Hereditas 58, 385--392. BUNg H. F. & DRYSI)ALE J. W. (1971) The separation of partially oxidized hemoglobins. Biochim. hiophys. Acta 229, 51. BUTCHER P. D. & HAWKEr C. M. (1977) Comparative study of haemoglobins from the Artiodactyla by isoelectric focusing. Comp. Bioch. Physiol. 56B, 335-339. CLEGG J. B. (1970) Horse hemoglobin polymorphism: evidence for two-linked, non-allelic or-chain genes. Proc. R. Soc. B. 176, 235-246. CLEGG J. B. (1974) Horse hemoglobin polymorphism. Ann. N.Y. Acad. Sci. 241, 61-69. CLEGG J. B., NAUGHTON M. A. & WEATHERALL D. J. (1966) Abnormal human haemoglobins: Separation and characterization of the :t- and fl-chains by chromatography, and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok). J. molec. Biol. 19, 91-108. DRYSDALE J. W., RIGHETT1 P. & BUNN H. F. (1971) The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim. biophys. Acta 229, 42 50. JEPPSON J. O. & BERGLUNDS. (1972) Thin layer isoelectric focusing for haemoglobin screening and its application to haemoglobin Mahno. Clin. Chim. Acta 40, 153-158. KILMARTIN J. V. & CLEGG J. B. (1967) Amino acid replacements in horse haemoglobin. Nature, Lond. 222, 1277 1278. KITCHEN H., BORESON D., MALK1NS S. & BRETT I. (1976) Horse Hemoglobin. In Proc. First. lnt'l Syrup. on Equine Hematology, (Edited by KITCHEN H. & KREHBIEL J. D.) pp. 42-47. MOHR E. (1970) Das Urwildpferd. Revised edition. A Ziemsen Verlag, Wittenberg Lutherstadt. MONTE M., BEUZARD Y. & ROSA J. (1976) Mapping of several abnormal hemoglobins by horizontal polyacrylamide gel isoelectric focusing. Amer. J. elin. Path. 66, 753-759. RYDER O. A., EPEL N. C. & BENIRSCHKE K. (1978) Chromosome banding studies of the Equidae. Cytogenet. Cell Genet. 20, 323-350. SHORT R. V., CHANDLEY A. C., JONES R. C. ALLEN W. R. (1974) Meiosis in interspecific equine hybrids. If. The Przewalski's Horse/domestic horse hybrid. Cytogenet. Cell Genet. 13, 465 478. SIMPSON G. G. (1951). Horses. Oxford Univ. Press, N.Y. TROMMERSHAUSEN-SMITH A., RYDER O. A. St, SUZUKU Y. (1979) Blood typing studies of twelve Przewalski's horses. Intl. Zoo Yrbk. (In press). VOLE J. (1976) Studbook of Przewalskfs Horses. Prague Zoo, Prague.
0305-0491 79 0401-0305502.00/0
H E M O G L O B I N P O L Y M O R P H I S M IN EQUUS PRZEWALSKII A N D E. CABALLUS A N A L Y Z E D BY ISOELECTRIC F O C U S I N G OLIVER A.
RYDER
Zoological Society of San Diego, Research Department, PO Box 551, San Diego. California 92112 U.S.A.: ROBERT S. SPARKES a n d 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 Przewalski'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 phenotypes observed in a family of Norwegian trotting horses. 4. Hemoglobin genotypes of fifteen Przewalski's horses were determined and inheritance of hemoglobin 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 Mongolian 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 morphological and biochemical criteria have established the close similarity of these two species (Simpson, 1950: Trommershausen-Smith et al., 1979). Cytogenetic 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 chromosomes 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 hemoglobins detectable by starch gel electrophoresis (Kilmartin & 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 focusing techniques to compare the hemoglobins of PrzewalskFs 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 without resorting to amino acid composition analysis of separated tryptic peptides. Electrofocusing analysis was also able to accurately identify the rare hemoglobin variants found in Norwegian trotting horses by Braend (19671 and further characterized by Clegg (1970).
305
MATERIALS AND M E T H O D S
Blood samples from E. caballus were obtained from normal adult animals by venipuncture. Blood samples from E. przewalskii were obtained by venipuncture of adult animals which had been chemically immobilized for routine veterinary procedures. Isoelectric focusing was performed in thin polyacrylamide slab gels on a commercial apparatus (LKB Multi-
OLIVER A. RYDER et al.
306
I
2
3
4
5
6
7
8
9
I0
Fig. 1. Isoelectric focusing of hemoglobins from E. przewalskii and E. caballus. Packed red cell hemolysates were subjected to isoelectric focusing as described in Materials and Methods. Slot 1: human HbA: slot 2: E. caballus--Clydesdale (BI/BI): slot 3: E. caballus--Shetland pony (BI/BI): slot 4: E. przewalskii "Bogatka" (BI/BII); slot 5: E. przewalskii "Henrietta" (BII/BII); slot 6: przewalskii "Jeanhold" (BII/BII); slot 7: E. przewalskii "Borkas" (BI/BII); slot 8: E. przewalskii "Bolinda" (BI/BI); slot 9: E. przewalskii "Roland" (BI/BII); slot 10: E. caballus--American standard breed (BI/BII). Breed designations are noted for E. caballus individuals; house names are given for E. przewalskii individuals. Hemoglobin genotypes (Clegg, 1970) as determined by electrofocusing are given in parentheses.
phor 2177). The gel was prepared according to the procedure recommended by the manufacturer (LKB Application Note 75, March 29, 1973) from the following reagents: 7.5g sucrose in 36.6ml H20, 10ml 29.17/o acrylamide (Eastman), 10ml 0.9°4, N,N'-methylene bisacrylamide (Eastman), 0.05ml N,N,N',N'-tetramethylethylene diamine (Eastman), 3.0ml pH 6-8 ampholine (LKB) 40% w/v, and 2.0ml 1% ammonium persulfate (Eastman). Twenty pl of a packed red cell hemolysate was added to 50/~1 of 0.1°/,, aqueous KCN. Consistent with the findings of Bunn & Drysdale (1971), it was observed that treatment of hemolysates with KCN was necessary to prevent extra bands. The KCN-treated hemoglobin solution was then absorbed onto 5 x 8 mm Whatman No. 3 filter paper and placed onto the gel approximately 2 cm from the cathodal Side of the gel. The electrophoresis apparatus was precooled with tap water for at least 30 min before the start of the run. Filter paper strips were saturated with i M H3PO 4 for the anode and 1 M NaOH for the cathode. The gel was run at constant current of 25ma for 30rain. At this time the filter paper which contained the hemolysate was removed. The run was then continued at 900 constant volts for 2 hr or until the current through the gel reached a constant value. Immediately after turning off the power supply, the gel was placed in 12.5~;, trichloroacetic acid for 5min, then rinsed in tap water. The gel was photographed and subsequently stored in water. For chemical analysis red cell lysates were converted to globin by the acid acetone method and the ~-fast,
~t-slow, and fl-chains were fractionated by chromatography on carboxymethyl cellulose in 8 M urea, 0.05 M 2-mercaptoethanol, pH 6.8 as described by Clegg et al. (1966). Separated ~t-fast and or-slow chains were digested with trypsin and the digests fractionated by high voltage electrophoresis on paper at pH 6.5 followed by chromatography in n-butanol: acetic acid: pyridine: water (15: 3: 10:12 by vol) (Kilmartin & Clegg, 1967). Chromatograms were developed by staining with 0.02%(, ninhydrin in acetone and heating at 60°C for 20 min. The ~4 peptides (which include residue 24) to be analyzed were eluted with 6 N HCI containing 2 mg/ml phenol to minimize destruction of tyrosine and hydrolyzed in sealed capillary tubes for 18 hr at 107°C. The hydrolysates were analyzed on an automated analyzer.
RESULTS AND DISCUSSION When KCN-treated packed red cell hemolysates were subjected to electrofocusing, hemoglobin patterns similar to those shown in Fig. l were obtained. Both E. przewalskii and E. caballus showed either two or four major hemoglobin bands. When just two hemoglobin bands were present they are quite well separated. When four hemoglobin bands are present the individual well separated bands are present as doublets (occasionally minor bands also occur even in KCN-treated samples).
Table 1. Correlation of E. przewalskii hemoglobin isoelectric focusing and tryptic peptide compositional analysis
Gel slot in Fig. 1
6 7 8
Species
E. przewalskii E. przewalskii E. przewalskii
Individual
Number of hemoglobin bands observed in electrofocusing
Jeanhold Borkas Bolinda
2 4 2
Amino acid Hemoglobin residue at genotype ~24 (Fig. 3) phe tyr/phe tyr
BII/BII BI/BII BI/BI
Electrofocusing of horse hemoglobins
pH8 $
pH6 ~
,, slow
afast
/ , a e o lys az4
A
tyr
~aso
lys =24 phe
/aso ~aso
gin az4 tyr
gin az4 I~e
307
60
8~
(;If 24 'y'
I
40 af
24 tyr
60
~i
•,/24
I
40 ,",,
tyr
24 tyr
60
/ ' I f 24 ;)he
I
40a,
24 phe
Fig. 2. Interpretation of isoelectric focusing patterns of E. przewalskii and E. caballus.
Fig. 3. Hemoglobin haplotypes in E. cabal/us (Clegg, 1970).
In order to determine whether the four hemoglobin bands detectable by electrofocusing correspond to the four known types of hemoglobin described by Kilmartin & Clegg (1967), analysis of the amino acid composition of purified s-chain tryptic peptides was performed. These results are summarized in Table 1. The Przewalski's horse individuals with two hemoglobin bands exhibited a single residue at position 24 of their ~-chains, having either tyrosine only or phenylalanineonly at this position. There is a clear difference in the isoelectric points reached by the electrofocused fast or slow hemoglobin molecules depending on whether tyrosine or phenylalanine is present (Fig. 1). An individual with four hemoglobin bands was found to have both tyrosine and phenylalanine present at ct24. Furthermore, the isoelectric points reached by four E. przewalskii hemoglobin forms were indistinguishable from those of E. caballus hemoglobins (Fig. 1). Analysis of the remaining tryptic peptide fragments of the or-fast and or-slow chains and from fl-chains in E. przewalskii disclosed no differences in composition from the corresponding peptides obtained from purified E. caballus ct and fl-chains. A diagrammatic interpretation of the electrofocusing and peptide analyses is presented in Fig. 2. The interpretation of the electrofocusing results presented in Fig. 2 was subjected to a further test by electrofocusing hemoglobins from E. caballus individuals with rare variant phenotypes. The unusual hemoglobin phenotypes of these Norwegian horses were first described by Braend (1967) and subsequently by Clegg (1970). They differ from the common pattern in that either the electrophoretically slow hemoglobin molecules are either present in reduced amounts or not at all. Clegg (1970) has suggested that three ~t-chain haplotypes may explain the observed phenotypes. The three postulated haplotypes would each consist of two
linked or-chain genes (Fig. 3). Two of the haplotypes would include genes coding for both lys and gin at ~60, and would code for either phe or tyr at ~t24 of both chains. The proportion of fast and slow hemoglobin bands is 60~o and 40~o respectively. The third haplotypes would contain only gin at ct60 and only tyr at ct24. Thus, if the diagram presented in Fig. 2 is correct, an A/BII pattern should have a 3-banded phenotype with the ~ band being a doublet. Similarly, an A/BI phenotype should be two banded, but the amount of ct~ hemoglobin should be approximately 50~0 of the amount present in a BI/BI homozygote. In Fig. 4, samples of cyanohemoglobin from A/BI and A/BII genotypes have been electrofocused in the same gel with hemoglobins from BI/BI and BII/BII individuals. The data obtained from electrofocusing analysis of the rare hemoglobin phenotypes are consistent with the representation in Fig. 2. Thus, isoelectric focusing of hemoglobins from either E. przewalskii or E. caballus may be used to determine the hemoglobin genotypes without resorting to purification of peptides from globin chains and subsequent amino acid analysis. The patterns obtained upon isoelectric focusing are reproducible and similar patterns may be obtained from fresh samples and samples which have been stored frozen for extending periods. Treatment with KCN before electrofocusing is an important step; incomplete conversion of all hemoglobin molecules present to their cyanohemoglobin forms results in multiple bands due to the differing Oxidation states of the heme iron (Bunn & Drysdale, 1971). The hemoglobin genotypes of fifteen Przewalski's horses from the San Diego Zoo and San Diego Wild Animal Park have been determined by isoelectric focusing (Table II). Inheritance of the hemoglobin haplotypes (Clegg, 1970) in E. przewalskii can be demonstrated through these data. For example, the
I
2
3
4
5
6
7
Fig. 4. Isoelectric focusing of hemoglobins from Norwegian trotting horses with unusual hemoglobin variants. Red cell hemolysates were subjected to isoelectric focusing as described in Materials and Methods. Slot 1: E. przewalskii "Borkas" (BI/BII); slot 2: E. caballus "Lisa" (A/BII); slot 3: E. przewalskii "Bolinda" (BI/BI); slot 4: E. caballus "Torfrid" (30~o ~f, 70~o ct~,phe only at ~t24); slot 5: E. przewalskii "Borkas" (BI/BII); slot 6: E. caballus "Tierra" (A/BI); slot 7: E. przewalskii "Jeanhold" (BII/BII). Hemoglobin genotypes (Clegg, 1970) as determined by direct chemical analysis are given in parenthesis.
308
OLIVER A. RYDER et al.
Table 2. Hemoglobin genotypes of fifteen Przewalski's horses House name Henrietta Bogatka Jeanhold Belkina Belzar Bolinda Bogdo Bellina Borkas Bektari Roxina Belaya Bonnette Roland Hermonia
Sex ~ ~ ,~ ; 3 , ~ ~ : = 5 i
House number WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP WAP CAT ~,
01 02 03 05 06 07 08 10 Il 12 13 14 15 26
Studbook Hemoglobin number genotype 268 504 469 603 638 406 473 319 685 678 480 458 339 320 737
BII/BII BI/BI1 BII/BII BII/BII BII/BII BI/BI BI/BI BI/BII BI/BII BI/BII BI/BI BI/Bll BI/BI BI/BII BI/BII
~'no house number.
individual, Borkas (BI/BII) is the offspring of Jeanhold (BII/BII) and Bolinda (BI/BI) (Table 2). It is, in fact, these three individuals whose hemoglobins are presented together in figure 1. Thus, the hemoglobin haplotypes postulated by Clegg (1970) for domestic horses, E. cahallus, also appear to be inherited in a Mendelian fashion in Przewalski's horse, E. przewalskii. The BI and BII haplotypes appear to be present in these animals at approximately equal frequencies, and the n u m b e r of heterozygotes present does not differ significantly from the proportion expected if the haplotypes are in Hardy-Weinberg equilibrium. Unfortunately, no data on the frequency of BI and BII haplotypes is available for sizable numbers of domestic horses. Therefore, a direct comparison between the relative haplotype frequencies in these two species is presently unknown. With the relative ease that the horse hemoglobin genotypes can be determined by isoelectric focusing, it should now be possible to compile data on haplotype frequency in E. caballus without the effort required for amino acid compositional analysis of tryptic peptides. Additionally, isoelectric focusing should enable hemoglobin genotypes to be used in parentage exclusion determinations in either of the two horse species examined here. As these studies were being conducted, our attention was brought to the report of Kitchen et al. (1976) which showed that the four different E. caballus hemoglobins could be demonstrated by isoelectric focusing.
Acknowledgements--We wish to thank the veterinary staff of the Zoological Society of San Diego, particularly Drs L. S. Nelson, J. E. Oosterhuis, P. T. Robinson and J. E. Meier for their cooperation in obtaining samples.
REFERENCES BRAEND M. (1967) Genetic variation in horse hemoglobin. Hereditas 58, 385--392. BUNg H. F. & DRYSI)ALE J. W. (1971) The separation of partially oxidized hemoglobins. Biochim. hiophys. Acta 229, 51. BUTCHER P. D. & HAWKEr C. M. (1977) Comparative study of haemoglobins from the Artiodactyla by isoelectric focusing. Comp. Bioch. Physiol. 56B, 335-339. CLEGG J. B. (1970) Horse hemoglobin polymorphism: evidence for two-linked, non-allelic or-chain genes. Proc. R. Soc. B. 176, 235-246. CLEGG J. B. (1974) Horse hemoglobin polymorphism. Ann. N.Y. Acad. Sci. 241, 61-69. CLEGG J. B., NAUGHTON M. A. & WEATHERALL D. J. (1966) Abnormal human haemoglobins: Separation and characterization of the :t- and fl-chains by chromatography, and the determination of two new variants, Hb Chesapeake and Hb J (Bangkok). J. molec. Biol. 19, 91-108. DRYSDALE J. W., RIGHETT1 P. & BUNN H. F. (1971) The separation of human and animal hemoglobins by isoelectric focusing in polyacrylamide gel. Biochim. biophys. Acta 229, 42 50. JEPPSON J. O. & BERGLUNDS. (1972) Thin layer isoelectric focusing for haemoglobin screening and its application to haemoglobin Mahno. Clin. Chim. Acta 40, 153-158. KILMARTIN J. V. & CLEGG J. B. (1967) Amino acid replacements in horse haemoglobin. Nature, Lond. 222, 1277 1278. KITCHEN H., BORESON D., MALK1NS S. & BRETT I. (1976) Horse Hemoglobin. In Proc. First. lnt'l Syrup. on Equine Hematology, (Edited by KITCHEN H. & KREHBIEL J. D.) pp. 42-47. MOHR E. (1970) Das Urwildpferd. Revised edition. A Ziemsen Verlag, Wittenberg Lutherstadt. MONTE M., BEUZARD Y. & ROSA J. (1976) Mapping of several abnormal hemoglobins by horizontal polyacrylamide gel isoelectric focusing. Amer. J. elin. Path. 66, 753-759. RYDER O. A., EPEL N. C. & BENIRSCHKE K. (1978) Chromosome banding studies of the Equidae. Cytogenet. Cell Genet. 20, 323-350. SHORT R. V., CHANDLEY A. C., JONES R. C. ALLEN W. R. (1974) Meiosis in interspecific equine hybrids. If. The Przewalski's Horse/domestic horse hybrid. Cytogenet. Cell Genet. 13, 465 478. SIMPSON G. G. (1951). Horses. Oxford Univ. Press, N.Y. TROMMERSHAUSEN-SMITH A., RYDER O. A. St, SUZUKU Y. (1979) Blood typing studies of twelve Przewalski's horses. Intl. Zoo Yrbk. (In press). VOLE J. (1976) Studbook of Przewalskfs Horses. Prague Zoo, Prague.
