INHERITANCE OF A HEME-BINDING IN RABBITS

ALLAN A. GRUNDERZ Serology Laboratory, Department of Veterinary Microbiology, University of California, Dauis Received June 2, 1966

LECTROPHORETIC variants of serum which bind hemoglobin E have been referred to in man (SMITHIESand WALKER1956) and swine (KRISTJANSSON1961) as . IMLAH(1964) found that these same electrophoretic variants of swine could be demonstrated only if old homoglobin or alkaline hematin rather than fresh hemoglobin was added to swine sera prior to electrophoresis. Consequently IMLAH( 1964) renamed this polymorphic pro- tein “haem-binding .” The polymorphic serum protein of rabbits which is to be described, exhibits somewhat similar characteristics to the “haem-binding globulin” of swine and will be tentatively designated “heme-binding protein” (Hbp) for reasons which will be discussed later (see DISCUSSION). In a pioneer study using starch gel electrophoresis, SMITHIES(1 955) demon- strated three distinct patterns of haptoglobins in human sera. These patterns were found by SMITHIESand WALKER(1956) to be controlled by two autosomal alleles Hp‘ and Hpz. Later, SMITHIES,CONNELL and DIXON(1962) and CONNELL, DIXONand SMITHIES(1962) subjected purified to reductive cleavage and electrophoresis in a starch gel containing mercaptoethanol and 8111 urea at acid pH, and were able to show that haptoglobin consisted of an alpha and beta chain. Three alpha chains were found to be controlled by three alleles-Hp’”’, Hp”, and Hp2-which could form six possible genotypes, all distinguishable by the above procedure. Using this same procedure, NANCEand SMITHIES(1963) found unusual phenotypes in a Brazilian population which were explained by hypothesizing the alleles HPFF7Hpzss, and Hp2(FS)in place of HP. SHIM,LEE, and KANG(1965) have presented evidence for three alleles, named HpP”, Hpfi‘, and HpfiL,which control the inheritance of beta chains of human haptoglobins. GIBLETT(1964) has referred to 11 variant haptoglobin phenotypes of man in addition to the three types first described. Electrophoretic variants of haptoglobins have not been described in many other species. KRISTJANSSON(1961 ) described six phenotypes of haptoglobins in swine sera. The synthesis of these haptoglobins was shown to be under the control of three codominant alleles. A fourth allele and its corresponding haptoglobin was discovered by HESSELHOLT(1963). This system of haptoglobins was later re- ferred to as a system of “haem-binding ” (IMLAH1964) as mentioned

From a thesis submitted in partial fulfillment of the requmements for the Ph D degree at the Umversitj of ( allforma, Davis Present address Animal Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Ontario Ldnada

Genetics 54: 1085-1093 Nmember 1966 1086 A. A. GRUNDER above, or as “haematin-binding proteins” ( GRAETZER,HESSELHOLT, MOUSTGAARD and THYMANN1964). BLUMBERG[1960] found no serum haptoglobin poly- morphism, such as that found in man, in a study of four chimpanzees (Pan toglodytes), 170 monkeys (Macaca mulatta), or 34 baboons (Papio). This communication will present evidence for genetic control of a heme-binding protein polymorphism in rabbit sera. Starch gel electrophoresis was used to demonstrate the polymorphism.

MATERIALS AND METHODS

Sources of samples: Blood samples were obtained from two breeds of rabbits (New Zealand Whites and Californians) in the breeding populations maintained by the U.S. Department of Agriculture in Fontana, California, and from rabbits of mixed breeding (largely New Zealand Whites) maintained as a nonbreeding population by the Serology Laboratory. Preparation of sera: The Hbp of the serum was detected in gels by peroxidase activity of the hemoglobin, which was presumably attached to the Hbp. Therefore, red cells from one or more rabbits were collected in citrated saline solution (2% sodium citrate and 0.5% sodium chloride), washed three times with 0.9% sodium chloride, and lysed with 9 volumes of H,O. One drop (0.05 ml) of this lysate was added to 0.2 ml of serum. In order to demonstrate consistently per- oxidase activity in a maximum number of regions, it was necessary to heat the mixture of serum + lysate for 30 minutes at 56°C prior to electrophoresis; unheated serum + lysate mixtures gave unsatisfactory results. A modification of this procedure for preparing the mixture will be dis- cussed (see RESULTS). Electrophoresis: Horizontal starch gel electrophoresis was performed. The gel buffer and electrode solution were essentially the same as those described by KRISTJANSSON (1963). The electrode solution consisted of 0.30 M boric acid plus 0.10 M sodium hydroxide. The gel buffer (pH 7.5) was made of 0.004 M monohydrated citric acid plus 0.014 M Tris(hydroxymethy1)amino- methane. Each gel consisted of 29g of hydrolyzed starch (Connaught Medical Research Labora- tories, Toronto) in 250 ml of gel buffer. Gels were prepared in the manner described by KRIST- JANSSON (1960,1963). The methods for conducting electrophoresis were similar to those described by GRUNDER, SARTOREand STORMONT(1965). Serum + lysate samples were placed 4 cm from the cathode end of the gel at right angles to the long axis of the gel. Electrophoresis was begun by applying 150 volts for 15 minutes, after which time the paper inserts were removed. Electrophoresis was then continued, using 300 volts, until the borate boundary had migrated 9 cm from the insert line (origin). The total time for electrophoresis was about 2 hours. No cooling device was used, al- though the gel surface became quite warm. After electrophoresis, the gel was sliced into halves for staining purposes. Staining: The cut side of one gel slice was covered with a benzidine solution (SMITHIES1959) which consisted of 200 ml distilled H,O, 0.4 g benzidine, 1.0 ml glacial acetic acid, and 0.4 ml of 30% H,O,. Peroxidase activity was exhibited by the formation of blue zones for the first 20 min- utes, followed by a gradual change to brown and eventual loss of zone resolution. Preservation of zones was attained by washing the gel slice, after 20 minutes of staining, in a 1:l (v/v) solution of methanol and water as suggested by STORMONTand SUZUKI(1966). Occasionally, the second gel slice was stained with a 1’% Amido Black solution for 30 seconds and then was destained in a 5:5:1 (v/v/v) solution of methanol, water, and glacial acetic acid.

RESULTS Phenotypes: After subjecting serum + lysate samples to starch gel electro- phoresis and subsequently staining the gel with benzidine solution, peroxidase A HEME-BINDING PROTEIN IN RABBITS 1087

I +

II

m

Hb Origin

2-2 3-3 2-2 1-1 1-3 1-2 2-3

FIGURE].-A photograph of a grl, showing four regions of hrnzidine staining. I. 11. 111. and Hh. Thr pattrrn on the far left (2-2) is of a heated serum plus lysate samplr ancl, therefore, ex- hihits rrgion I. The remaining six patterns of mixtures of serum plus hratrtl lysate lack region I, hut exhihit Hhp phenotyprs 3-3.2-2. 1-1. 1-3. 1-2. and 2-3. Zones of region I1 have bern markrd with wtiitr clots. activity became evident in four regions. These were named I, 11. 111. and Hb in order of their proximity to the anode end of the gel (Figure 1). Region I was located in the area. This region occasionally contained two closely spaced zones. but no individual variation was observed with regard to migration within this region. The zones of region I appeared only if the serum -!- lysate mixture was heated to 56°C for 30 minutes (see Table 1. also Figure 1). Region I1 was located in the area behind the zone (not shown), a zone which appears upon staining the gel with Amido Black and which contains a protein with the ability to bind Fe"" (unpublished). It was observed early in this investigation that zones appeared in region I1 upon staining with benzidine solution only if the serum + lysate samples had been heated prior to electro- phoresis. It was found subsequently that, when lysates alone were heated to 56°C for 30 minutes and then mixed with unheated serum, zones with good peroxidase activity appeared in region 11. An unheated mixture of 3-month-old serum and fresh lysate sometimes produced acceptable patterns in region 11. Results (Table 1088 A. A. GRUNDER TABLE 1 Required conditions for the appearance of various regions of benzidine staining

Appearance* of regions Materials subiected to electroDhoresis I I1 I11 Hb Fresh+ serum 00 ?O Fresh lysate 00o+ Fresh serum + Fresh lysate 00++ Fresh serum (56°C)+ Fresh lysate 00++ Fresh serum + Fresh lysate (56°C) o+ ++ (Fresh serum + Fresh lysate) (56°C) ++ ++ 3-mo-old serum + Fresh lysate O?++ 3-mo-oldserum + Fresh lysate (56°C) o+ ++

* +=noma1 staining, using (Fresh serum + Fresh lysate) (56OC) as the standard; O=no staining; ?=occasional light staining. + Fresh means less than 24 hours from time of collection of serum or cells for lysates to the time of electrophoresis.

1) of attempts to obtain optimal resolution of region I1 indicated that lysates must be heated to 56°C for 30 minutes, while heating or aging of serum seemed unimportant. It was suspected that the heat decomposed the hemoglobin into heme plus globin and that region I1 represented a complex of serum protein plus heme which permitted the peroxidase reaction. Hence, this serum protein was tentatively named heme-binding protein (Hbp) . There appeared to be five major zones in region I1 (Figure I).Minor zones of region 11, which are more obvious on the anode side of the first four than on the last three patterns of Figure 1, were not considered in this study. The various combinations of these five major zones designated a through e (Figure l),were revealed in six distinguishable phenotypes. These phenotypes were named Hbp 1-1, Hbp 2-2, Hbp 3-3, Hbp 1-3, Hbp 1-2, and Hbp 2-3, on the assumption that they are controlled by the three codominant alleles Hbp', Hbp2, and HbpS. Phenotype Hbp 1-1 was characterized by zones a and c, phenotype Hbp 2-2 by zones b and d, while phenotype Hbp 3-3 contained the slowest zone, e, and another which migrates at the same rate as the slower zone of Hbp 1-1, also called c. Thus these three phenotypes-the presumed homozygotes-were each characterized by two major zones stained to about equal intensity. Phenotype Hbp 1-3 contained zones a, c, and e, with c being heaviest in intensity and about equal to c of the corresponding homozygotes, Hbp 1-1 and Hbp 3-3. While phenotypes Hbp 1-2 and Hbp 2-3 could be distinguished fairly readily from the homozygotes, repeated runs were often necessary to distinguish these phenotypes from Hbp 1-3 and especially from each other because the zones within these two phenotypes were always lighter than corresponding zones of homozygotes and were not easily resolved (Figure 1). Hbp 1-2 possessed zones c and d, which seemed about equal in staining intensity, as well as a very light a zone, which was almost always lost in a diffuse staining environment. Similarly, Hbp 2-3 con- tained zones d and e, which were of about equal intensity, as well as a very light zone b, which was difficult to discern. Zones b and c could not be resolved in Hbp A HEME-BINDING PROTEIN IN RABBITS 1089 1-2 and Hbp 2-3, respectively, probably because of their proximity to other zones. In uitro mixtures of serum + lysate samples of phenotypes Hbp 1-1 + Hbp 3-3, Hbp 1-1 + Hbp 2-2, and Hbp 2-2 f Hbp 3-3 produced approximately the same benzidine-stained gel patterns as those produced by serum f lysate samples of phenotypes Hbp 1-3, Hbp 1-2, and Hbp 2-3, respectively. Occasionally one or two very light staining zones in addition to the minor zones already mentioned, were observed to migrate anodally to the six phenotypes of region 11. These were more prevalent when older serum f lysate mixtures were subjected to starch gel electrophoresis, or when the serum f lysate mixtures were heated to 56°C for 30 minutes prior to electrophoresis, than when lysates were heated separately and were then added to unheated sera and subjected to starch gel electrophoresis. The latter method of preparing serum + lysate mixtures was preferred, in order to avoid these extra confusing zones. The protein stain, Amido Black, revealed similar zones in terms of number and position to those of region I1 found with the bezidine stain. However, the patterns of zones on the protein-stained gel were extremely difficult to interpret because of background staining, and thus gels were stained routinely only with benzidine. Region I11 consisted of a single zone and could be detected in patterns of un- heated serum + lysate mixtures (see Table 1 for other methods). When serum alone was subjected to starch gel electrophoresis, region I11 as well as a zone be- tween regions I1 and I11 sometimes stained faintly with benzidine solution. How- ever, investigation of the nature of benzidine stained zones of sera as well as some apparent variation in intensity and migration rate of the zone of region I11 of serum f lysate mixtures (see Figure 1) was not pursued. Hemoglobin designated Hb in Figure 1 appeared near the origin. This hemo- globin was probably unbound to any serum component because it migrated at the same rate as hemoglobin of lysates that were not mixed with sera. No individual variation was observed in this Hb region. Family data: Soon after commencement of the Hbp study, it was observed that the progeny, normally 4 weeks of age at the time of bleeding, exhibited a zone pattern in reigon I1 which was much less intensely stained than that of adults (older than 6 months). It was especially difficult to diagnose phenotypes Hbp 1-3. Hbp 1-2, and Hbp 2-3 with certainty in these young rabbits. Mature litters were not available for parent-progeny analysis. Of the 49 4-week-old progeny tested for Hbp, as recorded in Table 2, 27 were typed while the remaining 22 could not be classified. However, only those Hbp phenotypes which were expected appeared in the progeny, expectation being based on the hypothesis that the six observed phenotypes are controlled by three codominant autosomal alleles, Hbp’, Hbp2. and Hbp’. Population data: The estimates of the allelic frequencies for three adult popula- tions are presented in Table 3. Only one member of a sib family, chosen at ran- dom, was included in the gene frequency survey of New Zealand Whites and Californians, in order to avoid the influence of variable family size on allelic fre- quency. Table 3 indicates that the New Zealand White population of the Fon- 1090 A. A. GRUNDER TABLE 2 Distribution of Hbp phenotypes in progeny from seven types of matings

No. of progeny of phenotypes Matings No. of litters Hbp 1-1 Hbp 2-2 Hbp 3-3 Hbp 1-2 Hbp 1-3 Hbp 2-3 Unclassified 1-1 x 1-2 2 6 0 0 0 0 0 5 2-2 x 2-2 1 0 6 0 0 0 0 0 3-3 x 3-3 1 0 0 3 0 0 0 0 1-2 x 2-2 1 0 1 0 0 0 0 7 1-1 x 3-3 1 0 0 0 0 2 0 7 1-2 x 3-3 1 0 0 0 0 2 0 3 1-3 X 3-3 1 0 0 6 0 1 0 0 tana Station lacked allele Hbp, while the frequency of this allele in the Serology Laboratory population, largely New Zealand Whites, was 0.16. In contrast to the low frequency of Hbpz in these two populations, the frequency of Hbpz was 0.57 in Californians, the most frequent allele in that population.

DISCUSSION Early attempts to demonstrate peroxidase activity in region I1 were unsatis- factory until the routine heating of the serum 4- lysate solutions or, more re- cently, the use of mixtures of unheated serum plus heated lysate was initiated. Such treatment is not necessary to demonstrate haptoglobins in man (SMITHIES 1955, 1959). Although KRISTJANSSON(1961) demonstrated haptoglobin pheno- types in swine without heating, HESSELHOLT(1963) routinely heated the hemo- globin (chicken) + serum mixture to 37°C for 30 minutes, while IMLAH(1964) could demonstrate this same polymorphism only if he used old swine hemoglobin or alkaline hematin. A P-globulin of human serum has been shown (SCHULTZE, HEIDEand HAUPT1961) to bind hemin but not horse hemoglobin or cytochrome c. The name has been suggested for this heme-binding protein of human serum by GRABAR,VAUX ST. CYR and CLEVE(1960), and later by SCHULTZEet al. (1961). Investigations, which were conducted subsequent to the present study, have shown that the serum protein of region I1 binds hematin and hemin but not hemoglobin. Therefore this serum protein may be described as a heme-binding protein. The ability of region I11 to bind fresh hemoglobin would indicate that the

TABLE 3 Frequency of alleles in three adult populations

Frequency of alleles Populations Adults Nbp' Hbpz Hbp3 New Zealand White 9 0.44 0.0 0.56 Californian 7 0.29 0.57 0.14 Serology Laboratory 45 0.61 0.16 0.23 A HEME-BINDING PROTEIN IN RABBITS 1091 serum protein of this region could probably be designated as true haptoglobin. Re- gion I11 may correspond to a hemoglobin-haptoglobin region demonstrated by means of starch gel electrophoresis by MURRAYand CONNELL(1960) during their investigation of the elevation of rabbit-serum haptoglobin through expen- mental means. Region I differed from both I1 and I11 in that it appeared only in gel patterns of serum samples heated together with lysates (Table 1). Apparently heat was necessary to promote the proper conditions for the combination of hemoglobin with serum protein in order to exhibit region I. Perhaps the serum protein in- volved in region I is albumin because region I and albumin are found closest to the anode in benzidine and Amido-Black-stained gels respectively. The lack of readily demonstrable Hbp in region I1 in 4-week-old rabbits as compared to relatively greater quantities in adults brings up the question of time of development for this protein. SMITHIES(1955) reported that haptoglobin was not always present in young children. From this observation in man, and that made in rabbits in this study, it would appear that serum haptoglobin and region I1 Hbp levels are normally low in the young, but gradually increase until a plateau is reached in adults of these two species. Also, it was thought that the amount of protein in region I11 was slightly reduced in 4-week-old rabbits com- pared to adults, although the amount of reduction was difficult to gauge since, as observed in adults (see Figure 1) , the amount of region I11 varied among sera of young rabbits. If one accepts the hypothesis that these phenotypes of region I1 are genetically controlled, there remains the problem of how two identical Hbp alleles produce two different zones. For example, how does an animal with genotype Hbp'Hbp' produce zones a and c in phenotype Hbp 1-1 (Figure l)?A simple hypothesis might be that the faster zone a is a single polypeptide or monomeric product of the allele Hbp' and that this monomeric unit polymerizes to form a dimer a-a which is larger and therefore more slowly migrating, being represented by the zone at c. This hypothesis may be easily extended to explain the gene products of the other two alleles but would have to be tested by means of physical-chemical measurements of the various zones of Hbp. Other explanations of the two zones are, however, conceivable. There is also the problem of why three instead of four zones were observed in Hbp phenotypes 1-2, 2-3, and perhaps 1-3, although 1-3 apparently possessed zone c which was observed in 1-1 and 3-3. Perhaps the gel system employed does not separate certain zones which migrate very close to each other, as suggested by BEARN,BOWMAN and KITCHIN(1964) to explain the observance of three zones instead of four for the heterozygous phenotype 2-1 of the human serum group-specific component (Gc) .

The author acknowledges the advice and encouragement of PROFESSORSC. STORMONT, W. C. ROLLINSand R. E. FEENEYduring the course of this investigation. A large part of the data was obtained from research conducted in cooperation with the U.S. Rabbit Experiment Station, Sheep and Fur Animal Research Branch, Animal Husbandry Research Division, ARS, U.S. Department of Agriculture, Fontana, California. 1092 A. A. GRUNDER

SUMMARY Four regions of peroxidase activity, named I, 11, 111, and Hb, were evident when samples of rabbit serum mixed with lysate were heated and then subjected to starch gel electrophoresis. Regions I and Hb were the same for all rabbits, while I11 showed quantitative and small migration rate differences, but these re- gions were not studied in detail. A study of region 11, tentatively designated as a region of heme-binding protein (Hbp), revealed six distinguishable phenotypes, named Hbp 1-1, Hbp 2-2, Hbp 3-3, Hbp 1-2, Hbp 1-3, and Hbp 2-3. These pheno- types were explained on the basis of control by three codominant autosomal al- leles, Hbp', Hbf, and HbpJ, but only limited population and family data were available to test this explanation. Hbp concentration in sera of several 4-week-old animals was lower than in adults such that Hbp classification of some offspring during family studies was precluded.

LITERATURE CITED

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