Comp. Biochem. PhysioL, 1975, VoL 51B, pp. 93 to 97. Pergamon Press. Printed in Great Britain

STRUCTURAL CHARACTERIZATION OF POLYMERIC HAPTOGLOBIN FROM GOATS

JAMES C. TRAVIS 1, JUDY GARZA ~ AND BOB G. SANDERS s

~Department of Biology, University of North Carolina, Charlotte, North Carolina 28213; and 2Department of Zoology, University of Texas, Austin, Texas, U.S.A.

(Received 6 March 1974)

Abstrnct--1. Polymeric haptoglobin was purified from the serum of Spanish goats (Capra hircus). 2. Subunits were demonstrated and purified for assay by column chromatography. 3. A large polypeptide subunit, comparable to beta from human haptoglobin, was demonstrated. 4. A second, smaller subunit exhibited properties similar to the alpha subunits from human hapto-

globin. 5. These data are discussed with respect to the possible evolutionary relationships between goat and

human haptoglobins and the genetic mechanisms which generated goat haptoglobin.

INTRODUCTION

TI-Ir~E rOWS of haptoglobin, Hps 1, 2-1 and 2, have been determined electrophoretically in the human population (Smithies & Walker, 1956). This polymorphism is due to the presence of two alleles at the Hp o~ locus, Hp a ~ and Hp a s, which code for the ¢~ and od polypeptide chains (Connell et al., 1962). The Hp fl locus is not allelic, but its product, the fl subunit, is shared by all three Hp types (Cleve et aL, 1967). Electrophoretically Hp 1 exhibits a single band, while Hps 2-1 and 2 exhibit multiple banding on gels which exert a seiving effect such as starch or polyacrylamide. Apparently the a s polypeptide initiates polymerization, resulting, in a series of Hp molecules of increasing size.

The human a 2 subunit exhibits a molecular weight approximately twice that of a ~ (Connell et aL, 1966), thus prompting the suggestion that the Hp a s allele arose from a partial gene duplication of the Hp a ~ allele. Structural evidence has substantiated this postulate (Smithies et aL, 1962; Black & Dixon, 1968).

Until recently no polymeric forms of Hp had been observed in species other than man. However, polymeric l ips in goats (Travis et aL, 1970), sheep (Jarrett, 1972; Travis & Sanders, 1972) and cattle (Goodger, 1972) have now been demonstrated. These observations suggest that the Hps of these Artiodactyla contain an aS-like subunit which promotes polymerization. Presumably then, the Hps of other mammalian species which exhibit a singlebanded electrophoretic pattern similar to human Hp 1, contain the more primitive at-like allele. These observations have prompted the postulation that a genetic event similar to but independent from that occurring in human Hp evolution, a partial gene duplication of the precursor

Hp a 1 allele, occurred in Artiodactyla evolution (Travis & Sanders, 1972).

The objective of the present study is to examine the hypothesis that the polymeric form of goat hapto- globin arose through a partial gene duplication of the Hp a 1 allele. This hypothesis will be examined by performing structural characterization studies on purified haptoglobin subunits obtained from goat sera.

MATERIALS AND METHODS

Two adult female Spanish goats were injected sub- cutaneously with 2.5 ml of turpentine in order to elevate circulating levels of Hp. Blood was taken by jugular puncture 48 hr later, with 10% EDTA used as an anti- coagulant (0.1 ml EDTA/10 ml blood). After centriguga- tion of the blood at 3000 rev/min the plasma was decanted and stored at +4°C until utilized. Hemolysis was minimal with this method.

Purified Hp was prepared by using a combination of salt precipitation, gel filtration chromatography and ion- exchange chromatography. An equal volume of saturated (NH4)sSO4 was added to plasma and, after stirring at +4°C overnight, the preparation was centrifuged and the precipitate dissolved in borate-saline, pH 8-3. The precipitated proteins were then chromatographed on a Sephadex G-200 gel filtration column (3.5x135cm). The void volume (Hp rich) peak was concentrated by pre-evaporation and submitted to DEAE-Sephadex chromatography with a pH 6.9 sodium-phosphate buffer. A molarity gradient from 0.01 to 1.0 M sodium- phosphate was utilized.

Locating Hp-rich fractions and testing for purity were accomplished by immunoelectrophoresis (Scheideger, 1955) using anti-goat plasma sera as previously described (Travis & Sanders, 1972).

In order to determine whether goat polymeric Hp exhibited subunit structure, lyophilized Hp was reduced and alkylated (Hirs, 1967) in 7 M guanidine hydro-

93

94 JAMES C. TRAVIS, JUDY GARZA AND BOB G. SANDERS

Table 1. Carbohydrate content of goat haptoglobins

Adjusted mol. wt. of Per cent g CHO/mole polypeptide

Intact protein 19'-23t - - - - Subunit 1 16I'-20" 6200-7800:1: 31,200-32,800 Subunit 2 24[-30* 4500-5500§ 13,000-14,000

* Experimental values. "[ Computed values assuming equimolar ratios of the two subunits. J; Based on a molecular weight of 39,000. § Based on a molecular weight of 18,500.

chloride, then eluted with 5 M guanidine hydrochloride through Sephadex G-200 (2 x 75 cm). A second sample was amino-ethylated (Cole, 1967) in 10 M urea/0"2 M Tris and eluted with 1 N acetic acid through G-200.

Molecular weight estimations of subunits were accomplished by gel filtration chromatography (Andrews, 1964). To test for purity and to compare electrophoretic mobilities of goat Hp subunits with those of human Hp, acrylamide gel electrophoresis with an acid-urea buffer, pH 4"0, was carried out (Jordan & Raymond, 1967). Carbohydrate determinations were accomplished utilizing the anthrone assay (Williams & Chase, 1968) and peptide maps were prepared after the method of Katz et al. (1959). Amino acid compositions were determined after acid hydrolysis at I10°C for 20 hr on a Beckman Model 120C automatic analyzer.

RESULTS

Figure 1 depicts the electrophoretic properties of goat polymeric Hp as compared to human Hps 1 and 2. From goat plasma which exhibited this pattern, pure Hp was obtained by the methods described. Figure 2 depicts the immunoelectrophoretic demonstrations of Hp purity from progressive protein fractionations. Approximately 50mg of pure Hp was obtained from 250 ml of goat plasma.

The gel filtration profiles for reduced and alkylated haptoglobin as well as aminoethylated haptoglobin were identical (Fig. 3). Peaks 2 and 3 were inter- preted as being separated subunits. Non-treated Hp revealed a single peak in the eluted volume.

Calibration of the gel filtration column with proteins of known molecular weights (Fig. 3) allowed molecular weight estimations of the sub- units. Figure 4 shows the curve obtained when the eluted volumes of the calibration proteins were plotted against the molecular weights. Peaks 2 and 3 (these shall be referred to as subunits 1 and 2, respectively), when quantitated from the calibration curve, exhibited molecular weights of 39,000 and 18,500, respectively.

The electrophoretic mobilities of the goat Hp subunits were compared to those of the human Hps. As can be seen in Fig. 5, goat subunit 1 has a mobility identical to human Hp/3, while goat sub- unit 2 exhibits a mobility intermediate between human l ip fl and Hp cal. No trace of cross contami- nation was observed between goat subunits 1 and 2 with this technique.

The carbohydrate content of intact goat Hp as well as the isolated subunits is presented in Table 1. Two values are presented in each case since complete agreement between the value of the intact protein and the sum of the subunits (assuming equi-molar ratios) was not obtained. The experimental values are the average of two assays which exhibited good precision. Apparently CHO had been inadvertently introduced into the system with subunit purification or the assays with the intact protein did not detect all of the CHO. In any event the range defined by the two values in each case probably covers the true values for the CHO contents of the proteins. The molecular weights of the protein moieties of the subunits are also presented in Table 1, approximately 32,000 for subunit 1 and 13,500 for subunit 2.

Table 2 presents the amino acid compositions of goat Hp subunits. Particular note should be taken of the cysteine content of each subunit and the

Table 2. Amino acid composition of goat haptoglobin subunits*

Subunit 1 Subunit 2

Cys 5.1 (0.016) 2.3 (0.017) Asp 33 (0.110) 15 (0.120) Tur 16 (0-053) 8 (0"066) Ser 23 (0"076) 11 (0-085) Glu 44 (0"150) 12 (0.097) Pro 13 (0.043) 6 (0"049) Gly 31 (0"105) 11 (0.087) Ala 21 (0"072) 9 (0-071) Val 25 (0"086) 12 (0.098) Ile 10 (0-034) 5 (O.039) Leu 15 (0-051) 10 (0.077) Tyr 15 (0.051) 5 (0.037) Phe 8 (0.026) 3 (0.026) Lys 24 (0-081) 9 (0.073) His 5 (0.016) 3 (0.022) Arg 10 (0.032) 4 (0.031)

* Values represent amino acid residues per mole of polypeptide based on molecular weights of 32,000 for subunit I and 13,500 for subunit 2. Numbers in paren- theses represent the per cent molecular wt that each residue contributes to the intact polypeptide. Samples (0.0025/~mole) were performic acid oxidized, then hydrolyzed for 20 hr (HC1, II0°C); analysis was carried out by automatic column chromatography.

,rT,x

H

Orig

Ca) (:b) (c) Fig. 1. The electrophoretic Hp patterns of (a) human 1, (b) human 2, and (c) goat sera. Hb represents free hemoglobin, Hp the haptoglobin-hemoglobin complex. The gel was 5.0% Cyanogum--41; 0"5 ml N,N,N*,Nl-tetramethylenediamine and 0-1 g ammonium persulfate/150 ml buffer; 4 mm thick. The buffer contained 0.04 M gly and 0"15 M Tris, pH 9.2. Sample consisted of serum, Hb and 10~ sucrose ( 5 : 2 : 3 ) ; 10 h applied. Electrophoresis was carried out for 90min, 500 V, 60 mA. Stain was 3,3-dimethoxybenzidine, 0"4 g, in 200 ml 5~ acetic acid, to which 0.2 ml of 30~ hydrogen peroxide

was added prior to applying to gel.

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Fig. 2. Immunoelectrophoretic patterns illustrating successive purification of goat Hp after CA) 50~o ammonium sulfate precipitation of goat plasma, (B) Sephadex G-200 chromatography of the pre- cipitate from (A) and (C) DEAE-Sephadex chromatography of the void volume peak from (B). The arrow indicates the Hp band in each panel. The two wells on each slide contained identical material. The antiserum placed into each trough was rabbit anti-goat plasma serum. The Hp solution used in slide (C) was prepared from lyophilized protein at a concentration of 1-5 mg/100 ml

buffer. Electrophoresis was performed for 2 hr at 8 mA/slide.

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Origin )

Fig. 6. Peptide maps of (A) goat Hi:) subunit 2 and (B) subunit I. Chromatography was performed along the horizontal with electrophoresis along the vertical. Two mg of trypsin digested material

was applied in each case.

Structural characterization of polymeric haptoglobin from goats 95

number of lysine and arginine residues, the sum of which indicates the expected number of tryptic digest peptides, assuming no extensive internal homology due to repeated amino acid sequence.

Tryptic peptide maps of the goat Hp subunits are presented in Fig. 6. Subunit 1 exhibits approxi- mately thirty-three distinct peptides, while subunit 2

exhibits approximately sixteen peptides. No common peptides are shared by the two subunits.

DISCUSSION The demonstration of polymeric Hp in the

Attiodactyla has suggested the idea that a genetic event similar to that which occurred in human Hp

I I I I I I

I x 10 6 5x10 4 2.3x l0 4 1.Tx10 4 mw mw mw mw

E 0 0 . 4

c~ 0-2 6

0"1

l 75 100 125 t50 175 200

V o l u m e , m l

Fig. 3. Chromatographic profile obtained by eluting reduced and alky]ated goat Hp through a Sephadex G-200 column, with 5 M guanidine-hydrochloride. Protein in collected fractions was located and quantitated with a Beckman DB-G spectrophotometer. The calibrations along the top of the graph indicate points of elution for four molecules (Dextran blue, human gamma heavy chain,

human light chain and human Hpa 2, from left to righ0 used to calibrate the column.

I0 j , j , I ' ' l ' I

x

4-

o~

o~ ~ n l t 2

%% " ,%

! , l r , 'I i , T T I I I00 150 200

Volume, ml

Fig. 4. Graph used to determine molecular weights of goat Hp subanits. The curve was constructed by plotting the elution points of three calibration molecules (Fig. 3) against their respective molecular

weights.

96 JAMES C. TRAVIS, JUDY GARZA AND BOB G. SANDERS

evolution, namely a partial gene duplication of the a 1 allele to give rise to the cd allele, has also occurred in Artiodactyla evolution. Specifically, it is postu- lated that both goat and human Hp systems have evolved from common fl and a~-like ancestral genes, and have acquired aS-like alleles through similar genetic mechanisms. The structural characteristics of goat Hp as presented in this report were determined in order to test this hypothesis.

The reduction of goat Hp yielded two subunit species, 1 and 2. Subunit 1 exhibited a molecular weight of 39,000, a CHO content of 20 per cent, and

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l l m l l

m m

(3 O r i g i n

a' a 2 ~ I 2

H u m a n Goat

Fig. 5. Electrophoretic properties of goat Hp subunits compared with those from human Hp. Gel was 6% Cyanogum-41 with an ascorbic acid-ferrous nitrate catalyst; buffer was acid-urea, pH 4.0. Electrophoresis was carded out at 500 V, 60 mA for 3 hr. Stain was 1% buffalo black in 10% acetic acid: destaining was per-

formed in 7% acetic acid.

a polypeptide molecular weight of 32,000. These values are very close to those of human fl, which has a molecular weight of 40,000, a CHO content of 25 per cent and a polypeptide molecular weight of 30,000 (Barnett et al., 1972). In addition, human fl and goat subunit 1 exhibit identical electrophoretic mobilities at an acid pH. Therefore, the idea that human fl and goat subunit 1 are the products of genes that share common ancestry appears favour- able.

If one postulates that human cd and goat subunit 2 are the products of genes that evolved from a common ~l-like ancestral gene, then certain characteristics should be predictable about the goat Hp system. If one assumes that the ancestral a 1 gene encoded for a protein of 9000 molecular weight, then the product of a partially duplicated allele (a~-like) should exhibit a polypeptide molecular weight between 9000 and 18,000. Human a ~ and goat subunit 2, with molecular weights of 17,300 and 13,500, respectively, satisfy this criterion. Human a I and a ~ are devoid of carbohydrate; therefore, it is assumed that the ancestral cd was devoid of carbohydrate and one would predict a goat subunit without carbohydrate. Goat subunit 2, however, contains a sizable carbohydrate moiety, approximately 30 per cent of the total molecular weight. To make this datum compatible with the hypothesis, one has to postulate a mechanism for

carbohydrate association in goat evolution, or assume that the ancestral a ~ contained carbohydrate, and this characteristic was lost in human evolution.

Human c~ ~ contains six cysteines, twice that for a 1. This presumably is the basis for the capability to initiate polymerization, since polymer formation is due to covalent bonding between cysteines. Goat subunit 2 contains two to three cysteines. This is less than the expected number, but does not preclude polymer formation. Goat polymeric Hp is pre- sumably formed through disulfide bonds, since reducing treatments liberated single polypeptides.

A further prediction of the duplication of genetic material is repeated amino acid sequence in the product. This has been demonstrated for human a 2 (Black & Dixon, 1968), and is reflected in tryptic peptide maps, in that twenty-five peptides would be expected, but approximately half that number is observed (Smithies et al., 1962) due to the internal homology. Goat subunit 2 contains two to three cysteines, nine lysines and four arginines, giving a theoretical yield of fifteen to sixteen tryptic peptides. This is the observed number (Fig. 6) which would indicate no extensive repeated amino acid sequence. However, a few appropriately placed substitutions would eliminate this effect with the fingerprinting assay.

In the human population both the a 1 and a + alleles have been maintained, providing the basis for polymorphism. No polymorphism has been ob- served electrophoretically in the Artiodactyla. Approximately 100 goats and sheep with detectable levels of Hp have been examined and only a single polymeric pattern has been observed. This would suggest the possibility of favourable selection for the allele initiating polymerization and/or selection against the precursor cd-like allele, assuming the stated hypothesis is true.

The above discussion clearly demonstrates that enough data are not available to defend strongly the position of a gene duplication in goat Hp evolution and common genetic ancestry with the human. While the data do not preclude this hypothesis, other alternatives are certainly tenable. For example, one could postulate that human and goat Hps contain fl subunits with common ancestry, but et subunits with independent evolutionary origins. Obviously more data, such as partial or complete sequence of goat subunits, are needed as a basis for further com- parisons.

Acknowledgements---This work was supported by N.I.H. Training Grant 2T01 GM00337-11 from the National Institute of General Medical Sciences, Welch Foundation Grant F-408 and N.I.H. AI09533-01A1.

REFERENCES

ANDREWS P. (1964) Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91, 222-233.

Structural characterization of polymeric haptoglobin from goats 97

BARNETt D. R., TONG-Ho L. & BOWMAN B. H. (1972). Amino acid sequence of the human haptoglobin fl chain--I. Amino- and carboxylterminal sequences. Biochemistry 11, 1189-1194.

BLACK J. A. & DIXON G. H. (1968) Amino acid sequence of the alpha chains of human haptoglobins and their possible relation to the immtmoglobulin light chains. Nature, Lond. 218, 736-741.

CImvE H., GORDON S., BOWMAN B. H. & BEARN A. G. (1967). Comparison of the tryptic peptides and amino acid composition of the beta polypeptide chains of the three common haptoglobin phenotypes. Am. J. Hum. Genet. 19, 713-721.

COLE R. D. (1967) S-Aminoethylation. Meth. Enzymol. 11, 315-317.

CONNELL G. E., DIXON G. H. & SMrrHIES O. (1962) Subdivision of the three common haptoglobin types based on "hidden" differences. Nature, Lond. 193, 505-506.

CONNELL G. E., S~rrH~s O. &DtXON G. H. (1966). Gene action in the human haptoglobins--II. Isolation and physical characterization of alpha polypeptide chains. J. molec. Biol. 21, 225-229.

GOOrmER B. V. (1972) Preliminary characterization of the bovine polymeric Hb binding protein and comparison of some properties with human haptoglobins. Aust. J. exp. Biol. Med. Sci. 50, 11-20.

HIRS C. H. W. (1967) Reduction and S-carboxymethyla- tion of proteins. Meth. Enzymol. 11, 199-206.

JAm~.ETr I. G. (1972) A polymeric form of haemoglobin- binding protein in sheep following metabolic and hormonal disturbance. Aust. J. biol. Sci. 25, 941-948.

JORDAN E. M. & RAYMOh'O S. (1967) New catalyst system for acid polyacrylamide gel. Supplement: E-C Bulletin, 4: 1.

KATZ A. M., DpmYER W. J. & ANFINSEN C. B. (1959) Peptide separation by two-dimensional chromatography and electrophoresis. J. biol Chem. 234, 2897-2900.

SCrIEmEC_,GER J. J. (1955) Line micro-methode de l'immuno-61ectropherese. Int. Arch. All. App. lmmunol. 7, 103-110.

SMITHIES O. & WALKER N. F. (1956) Notation for serum- protein groups and the genes controlling their in- heritance. Nature, Lend. 178, 694--695.

SMrrmES O., CONNELL G. E. & DIXON G. H. (1962) Inheritance of haptoglobin subtypes. Am. J. Hum. Genet. 14, 14-21.

TRA~S J. C., BROWN S. O. & SANDERS B. G. (1970) A polymeric form of haptoglobin in the gamma- irradiated Spanish goat. Biochem Genet. 4, 639-649.

TRAVIS J. C. & SANDERS B. G. (1972) Haptoglobin evolution: polymeric forms of Hp in the Bovine and Cervidae families. J. exp. Zool. 180, ~41-148.

WILLL~IS C. A. & CHASE M. W. (1968) Carbohydrate analysis. Meth. lmmunol. Immunochem. 2, 282-305.

Key Word Index--Goat; Capra hircus; haptoglobin; polymeric haptoglobin; evolution of haptoglobins; subunit structure; haptoglobin alleles.