Co,ll,. Biochcm. Phlsiol.. Vol. 02B, pp. 339 lo 344. ~, Pcrg;irnon Press Lid 1979 Printed in Great Brit~in
0305-0491 79'0401-0339502.00'0
CANINE HAPTOGLOBIN" A U N I Q U E HAPTOGLOBIN SUBUNIT ARRANGEMENT* ALEXANDER KUROSKYi', REGINE E. HAY and BARBARA H. BOWMAN Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550, U.S.A. (Received 7 August 1978)
Abstract--1. Isolated canine haptoglobin behaved identically to the Gt2fl2 structure typical of human haptoglobin type i-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptogiobin dissociated into otfl subunits that separated into • and fl chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine ~t chain when compared to human ct~ chain. 4. These results provide evidence that there is no inter ct chain disulphide in canine haptoglobin comparable to the 0tt20~tl20 disulphide in human haptoglobin that links the two ~tfl subunits.
INTRODUCTION
Haptoglobin is a glycoprotein found predominantly in the ~t2 fraction of serum. It is present in sera of all classes of vertebrates studied (Sutton, 1970; Putnam, 1975). The electrophoretic mobility of Hp§ from most animals resembles that of human Hp 1-1 after alkaline gel electrophoresis (Schwantes & Tondo, 1969; Kurosky et al., 1976b). Notable exceptions are some members of the order Artiodactyla (Travis & Sanders, 1972) and some species of monkey (Beckman & Cedermark, 1960) which demonstrate polymeric Hp analogous to human Hp 2-2 and 2--1. Shifrine & Stormont (1973) examined Hp in sera from 200 beagles and found no evidence of polymorphism or polymer formation. The mean concentration of Hp in the serum of healthy dogs is 1.04 mg/ml, similar to that reported for man (0.89 mg/ml)(Harvey, 1976). Like Hp from other species, canine Hp increases in concentration 4- to 10-fold during inflammation (Ganrot, 1973). The purification and partial characterization of canine haptoglobin have been reported (Kurosky et al., 1976b; Uspenskaya et al., 1967, 1968). In all species investigated that possess nonpolymeric Hp, the functional molecule is a tetrachain structure comprised of two ~t and two fl chains that are covalently linked by interchain disulphide bonds (Smithies et al., 1962a; Malchy & Dixon, 1973a; Malchy et al., 1973; Kurosky et al., 1976a; Shim et al., 1971; Lockhart et al., 1972; Shim et al., 1975; Kong & Shim, 1974). A single disulphide bridge attaches the ~t and fl chains to form an 0~fl subunit in human Hp 1-1. Two such subunits are combined by an ct120-ct120 disulphide bond (Malchy & Dixon, 1973a; Kurosky et al., 1976a). The structure of human
Hp 1-1 is diagrammatically represented in Fig. 1. The one exception in interchain disulphide arrangement in animal Hp was described by Lee et al. (1974). In this report evidence was presented that rabbit Hp contained no interchain disulphides. In most species examined carbohydrate is found only on the hp /~ chain (20Yo in human hp fl chain), except for goat, which was reported to have carbohydrate on its :t chain as well (Travis et al., 1975). This paper compares the interchain disulphide arrangement of human, rat, rabbit, and canine Hp and presents evidence for an interchain disulphide arrangement in canine Hp that is unique among haptoglobins studied thus far. MATERIALS AND METHODS
Plasma was obtained from the following animals: (I) rabbit--an outbred strain of New Zealand Whites; (2) rat--an outbred strain of Sprague-Dawley; and (3) dog-greyhounds. Rat and rabbit plasma represented pooled samples but the plasma obtained from two dogs was processed separately, Pth amino acids and all reagents used in the sequencer were "Sequanal Grade" purchased from Pierce Chemical Co. and Beckman Instruments, Inc. Acrylamide and N,N'-methylenebisacrylamide, "Electrophoresis Purity", were products of Bio-Rad Laboratories. Carboxypeptidase A, treated with diisopropylfluorophosphate, was purchased from Worthington Biochemical Corp. Haptoglobin was isolated as previously described (Kurosky et al., 1976b) from plasma of rat, rabbit, and dog conditioned to acute phase inflammation by turpentine injection. All Hp purification steps were promptly carried out at 4 ° to minimize any possible protein alteration, and only freshly drawn plasma was used. Haptoglobin homogeneity was investigated by polyacrylamide gel electrophoresis (Woodward & Clark, 1967) and by SDS polyacrylamide gel electrophoresis (Weber & Osborne, 1969). * This investigation was supported by grant HD 03321 As previously reported (Kurosky et al., 1976b), the ct and from the National Institute of Child Health and Human fl chains of purified Hp were separated by gel filtration following reduction with 2-mercaptoethanol and alkylation Development. with iodoacetamide. Homogeneity of the isolated chains t To whom correspondence should be addressed. § Abbreviations: Hp, haptoglobin (intact); hp, haptoglo- was established by polyacrylamide gel electrophoresis and bin (chains); Hb, hemoglobin; Pth, phenylthiohydantoin; automated sequence analysis. Molecular weight estimates of intact canine Hp and SDS, sodium dodecyl sulphate; EDTA, ethylenediaminereduced and alkylated Hp were conducted essentially by tetracetic acid. 339
340
ALEXANDER KUROSKY, REGINE E. HAY and BARBARA H. BOWMAN
I05 I S
20 34 I I I
NI
r s--s7 Fs-s7 /~CHAIN 148 179,9o 2B z . ~ c
S
68 I 172 83 C or CHAINS
S
1 ] ' ~ - ~ " t 72 85 C 20 34 68 I
NI
S
I 5
N
[ 105
I
rsI~S7 148
F s-s7 245c ~ CHAIN 179 190 219
Fig. 1. Tetrachain arrangement of human Hp 1-1. Numbering of ct chain residues was revised (Kurosky et al., 1976a). Numbering and disulphide arrangement of fl chain residues were established by sequence analysis (Kurosky, Barnett, Lee, Touchstone and Bowman, in preparation). the methods of Weber & Osborne (1969), Swank & Munkres (1971), and Segrest et al. (1971), as we previously described (Kurosky et al., 1976a). After electrophoresis the gels were stained for protein with Coomassie brilliant blue R or for carbohydrate with the periodic acid-Schiff stain (Zacharias et al., 1969). Gels stained for carbohydrate were first washed overnight with 25% isopropanol/10% acetic acid and then with 10% isopropanol/10% acetic acid before staining. A molecular weight estimate of canine Hp was also made by gel filtration on a calibrated column of Sephadex G-100 (2.5 cm x 110 cm) equilibrated with 0.1% NH4HCO3, pH 8.5. Protein hydrolysis of the ct chain of canine Hp was carried out at 107° in 5.7 N HCI for compositional analysis and at 100° and 4 N HCI for hexosamine analysis. Methods of amino acid analysis were recently described (Kurosky et al., 1977). Quantitation of neutral sugars was made by the Winzler orcinol-sulfuric acid procedure (Fran-
cois et al., 1962) and of sialic acid by the thiobarbituric acid assay (Warren, 1959). Reaction with carboxypeptidase A was carried out according to Ambler (1972). Sequencer operation and methods of identification and quantitation of Pth amino acids were as previously detailed (Kurosky et al., 1976b). RESULTS Comparative acrylamide gel electrophoresis of rat, rabbit, dog, and human Hp indicated that the electrophoretic mobility at pH 8.9 was similar for all four species of Hp (Fig. 2a). In the presence of urea the electrophoretic mobilities of rat, rabbit, and human Hp were identical but the mobility of canine Hp was significantly faster (Fig. 2b). The elution volume of canine Hp on Sephadex G-100 corresponded to an !
~ii~ ~ ~i
: ii ¸¸¸
~
//
i
/
(+)
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li :::, !i:i:i: ......,. i:ii:
!
i
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i i~ii~i~!~i:~i¸
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!]iiI:
i~!~!i{iii~i!i!~i~i!!i!ii~!~i~i;i!~i~i~:~~i:~,~:i~i~ ¸ ~il~i~;~i:~!i~:ii~iil~
1 Hu
'!i: Do
Rt (a)
Origin
C-) Rb
Rb Rt
Do Hu (b)
Fig. 2. Slab polyacrylamide gel electrophoresis of human (Hu), dog (Do), rat (Rt), and rabbit (Rb) Hp. Gel buffer, 0.1 M tris-borate-EDTA-lactate, pH 8.9, 5% acrylamide (Woodworth & Clark, 1969). (a) without urea; (b) contained 6 M urea. 200pg of protein per slot, stained with Coomassie blue.
Canine haptoglobin subunit arrangement
341
Table 1. Comparison of the molecular weight of canine haptoglobin with rat, rabbit and human haptoglobin ' Canine
Rat
Rabbit
Human i-i
Observed
Corrected b
Observed
Corrected b
Observed
Corrected b
Observed
Sequence c
Intact Hpd
48,500
37,100
102,200
78,100
111,200
85,000
113,600
86,850
a Chaine
19,500
ii,i00
16,300
9,300
16,300
9,300
16,100
9,189
Chaine
33,900
29,400
35,300
30,600
38,500
33,300
39,500
34,200
Determined by SDS polyacrylamide gel electrophoresis according to Segrest et al. (1971). b Corrected relative to human Hp 1-1. c From primary structure analysis. d Nonreducing conditions in 5% acrylamide. Hp electrophoresis in 5%, 7.5%, 10%, and 12.5% gels containing 2-mercaptoethanol gave a minimum fl chain molecular weight (observed) at 12.5%. The ~ chain molecular weight was donstant and was averaged.
estimated molecular weight of 100,000 and was the same as human Hp 1-1. The observed molecular weights of the different Hp's and their component chains obtained by gel electrophoresis are summarized in Table 1. After SDS polyacrylamide gel electrophoresis, canine ~ and fl chains and human fl chain stained positively with the periodic acid-Schiff reagent but human ~ chain did not. The amino acid composition of carbamoylmethylated at chain of canine Hp is compared to human ~t chain in Table 2. Hydrolysis of canine ~t chain with carboxypeptidase A is shown in Fig. 3. Automated sequence analysis of the ~ chain of canine H p revealed the amino-terminus to be partially blocked due to cyclization of a glutaminyl residue giving rise to a pyrrolidonecarboxylyl residue. Since cyclization of the terminal glutamine was not complete, we were able to confirm nine of the amino-terminal residues as indicated in Table 3. Carbohydrate analysis of canine
ct chain indicated 3% neutral hexoses and 3% sialic acid. DISCUSSION In nondissociating buffers, canine Hp behaved similarly to human Hp during both polyacrylamide gel electrophoresis and gel filtration. However, in dissociating buffers, either urea or SDS, the protein migrated like an ~fl subunit rather than a n ~2fl2 tetrachain molecule. The slower mobility of rat Hp when compared to human Hp (Fig. 2a) was the result of aggregation as evidenced by the diffuse pattern of staining. In the presence of urea (Fig. 2b) the mobility of rat Hp was more like that of human Hp. In contrast to the report of Lee et al. (1974), rabbit Hp could not be separated into ~ and fl chains without the addition of a reducing agent. Heating rabbit Hp in the presence of I% SDS at 60 ° for two hr also
Table 2. Comparison of the amino acid compositions of canine ct and human :d chains of Hp" Canine a chain b
Lysine Histidine Arginine CM-cysteine d Aspartic acid Threonine d Serine d Glutamic acid Proline Glycine Alanine
7.8±.2 3.0±.I 1.2±.i 3.0±.I 8.6±.3 5.9±.I 2.8±.I 13.6±.3 6.9±.4 5.9±.2 2.8±.i
(8) (3) (i) (3) (8-9) (6) (3) (13-14) (7) (6) (3)
Human a I chain c
Amino Acid
8 2 2 4 14 3 2 9 7 7 5
Valine Methionine d Isoelucine Leucine Tyrosine Phenylalanine Tryptophan e Glucosamine f Galactosamine
Canine a chain b
7.7±.2 0.8±.1 1.9±.2 4.0±.2 3.7±.I 1.0±.03 1.1 4.0 0
Total Amino Acids
(8) (1) (2) (4) (4) (i) (i) (4)
82-84
Human al chain c
8 0 3 3 5 0 i 0 0 83
Values calculated from duplicate time-course hydrolyses: 24, 48, and 96 hr. b Residues per mol of protein based on the molecular weight of human ~1 chain (MW 9, 189). Residues established by sequence analysis (Black & Dixon, 1968; Malchy & Dixon, 1973b; Kurosky et al., 1976a). d Zero time of hydrolysis calculated from linear regression analysis. e Average of duplicate hydrolyses after reaction with mercaptoethanesulfonic acid (Penke et al., 1974). r Time-course hydrolysis for 6, 12, and 18 hr established maximum release to occur at 12 hr.
342
ALEXANDER KUROSKY, REGINE E. HAY a n d BARBARA H. BOWMAN 1
I
I
I
2
3
I
,
4
5
//
,
1.0c-~ la3
co
0.8-
h.I t.d
QO Q6~m b] Ou "~n-"
._1
~
0.2-
3~3
TIME (rain) Fig. 3. Carboxypeptidase A hydrolysis of carbamoylmethylated canine ~ chain at 37" (0) glutamine, (O1 valine; enzyme/substrate = 1/150 (w/w). (I) glutamine, ([]) valine, enzyme/substrate = 1/15 (w/w). Molecular weight of polypeptide portion determined to be 9,314 from compositional analysis (see Table 2j. Table 3. Comparison of the amino-terminal sequence of canine ~ chain with human ~ chain I
2
3
4
5
6
7
8
9
Human a
Val-Asn-Ser-Gly-Asn-Asp-Val-Thr-Asp /
Canine b
Gln-Asn-Thr-Gly-Ser-Gln-Ala-Thr-Ala/
Revised sequence, see Kurosky et al. (1976a). b Established by automated sequence analysis. Partially blocked (80%) by Gin cyclization. did not alter its mobility on SDS polyacrylamide gel electrophoresis. The observed molecular weights of the various animal Hp's and their corresponding chains from SDS polyacrylamide gel electrophoresis were corrected for anomalous mobility as shown in Table 1. These corrections were based on the molecular weight of human Hp calculated from compositional and sequence analysis (Kurosky et al., 1976a). The observed/3 chain values were about 12% exaggerated and intact Hp was elevated by about 22% due to carbohydrate attachment. Malchy et al. (1973) have also reported similar anomalous Hp mobility on SDS polyacrylamide gels. The electrophoretic mobility of human ~1 chain in SDS, as well as in SDS-urea polyacrylamide gels was consistently higher than expected based on the molecular weight determined by sequence analysis (Black & Dixon, 1968; Malchy & Dixon, 1973b; Kurosky et al., 1976a). The exact cause of this anomalous mobility is presently unclear. One possible explanation may be that the relatively high percentage of prolyl residues (8.4%) clustered at positions 17 and 78 restricts free rotation and gives rise to a more extended molecule. The occurrence of chain dimers was unlikely since prior heating of ~1 chain in 1% SDS and 1% 2-mercaptoethanol at 100°
for 10 min followed by 4 hr at 60° did not alter its mobility during SDS gel electrophoresis. The anomalous mobility of the hp al chain appears to be related to the gel electrophoretic system employed since ultracentrifugation studies gave a molecular weight of 8,860 + 400 (Smithies et al., 1962b). Anomalous mobility during polyacrylamide gel electrophoresis has been reported for a number of other proteins (Swank & Munkres, 1971; Banker & Cotman, 1972). The molecular weights of the • chains of rat and rabbit Hp are comparable to that of human (MW 9,189); however, the molecular weight of canine chain is comparatively larger as a result of carbohydrate attachment. Evidence that the molecular weight of the polypeptide portion of canine ~ chain is similar to human ~1 chain is supported by compositional analysis (Table 2) which gave nearly integral values and indicated 82 and 84 residues as compared to 83 residues for human a t chain. In addition, the molar release of carboxyl-terminal residues by carboxypeptidase A (Fig. 3) was in agreement with the polypeptide molecular weight calculated from compositional analysis (MW 9,314). Amino acid compositional analysis of the at chain of canine Hp indicated only three half-cystinyl residues compared to four reported for the cta chain of
Canine haptoglobin subunit arrangement human Hp. This result was consistent with the electrophoretic evidence and suggests that the half-cystinyl residue comparable to ct~20 in human Hp is replaced in canine ~ chain by some other amino acid. Unlike human ct chain, canine ~t chain contains a phenylalanyl and a methionyl residue. In addition, four residues of glucosamine were calculated per mol of canine :t chain which indicated carbohydrate attachment in agreement with the positive reaction obtained with the periodic acid-Schiff reagent. Automated sequence analysis of the amino terminus of canine ~t chain identified nine residues, six of which were different but chemically similar to comparable residues in human ct chain. Although this is only a small segment of ct chain, it is distinctly more variable than similar amino-terminal segments of canine and human fl chains (Kurosky et al., 1976b). Sequencer analysis of intact canine Hp indicated that the ~ chain was about 60~o blocked due to cyclization of its amino-terminal glutaminyl residue. The degree of cyclization increased to about 80% for purified ~t chain as a result of chemical manipulation during the isolation procedure. Hydrolysis of dog hp ct chain with carboxypeptidase A revealed a carboxyl-terminal sequence of valyl-glutamine identical to human ~t chain. However, the residue adjacent to valine must not be proline, as in the human, since carboxypeptidase A does not normally release residues preceded by proline. These results agree with the carboxyl-terminal evidence of dog Hp as reported by Uspenskaya et al. (1967). Investigation of the Hb binding capacity revealed that canine Hp was able to bind Hb as effectively as human Hp indicating that the inter :t chain disulphide linkage in canine Hp is not essential for Hb binding. Although the inter ct disulphide is absent, the ctfl subunit of canine Hp dimerizes to give a tetrachain structure, i.e., (ctfl)2, as judged by its electrophoretic mobility in nondissociating conditions (Fig. la) and by gel filtration on Sephadex G-100. The dimer is readily dissociated in the presence of urea or SDS. Interestingly, selective cleavage of the inter ~ disulphide in human Hp 1-1 by sulphitolysis does not disrupt its tetrachain structure as evidenced by electrophoresis in alkaline borate buffer (Malchy et al., 1973). Therefore, although a tetrachain structure is indicated for most animal haptoglobins investigated to date, it is apparent that other subunit or chain arrangements are also possible as indicated by the (~tfl)2 structure of canine Hp. Moreover, the human polymorphic Hp types 2-1 and 2-2, which give rise to a polymeric series covalently bonded by disulphide linkage (Fuller et al., 1973; Pastewka et al., 1975), are additional examples of variation of Hp subunit structure. All of these structural variations are capable of binding Hb. It is not uncommon that families of homologous proteins demonstrate multiple chain or subunit arrangements, e.g., the serine proteases. In view of the homology of Hp to the chymotrypsinogen family of serine proteases (Kurosky et al., 1974), it is worthwhile to point out that human clotting factor XIa is a tetrachain structure comprised of a pair of heavy chains and a pair of light chains held together by disulphide bonds (Kurachi & Davie, 1977). Haptoglobin from both dogs (greyhounds) used in
343
this study gave identical electrophoretic patterns on polyacrylamide gels. It will be of interest to examine the disulphide arrangement of Hp isolated from other breeds of dogs as well as from related families in order to define the extent of genetic variation of ct chain in the order Carnivora. SUMMARY
Electrophoretic characterization of canine Hp under dissociating conditions revealed a molecular weight corresponding to an ~tfl structure rather than an ct2fl2 chain arrangement identified for most nonpolymeric animal Hp. Compositional analysis of canine :t chain indicated only three half-cystinyl residues which, together with the electrophoretic evidence, suggested that the inter ~t chain disulphide comparable to Cys-20-Cys-20 in human ct~ chain is absent. The ~tfl subunit of canine Hp occurred as a dimer, (~tfl)2, under nondissociating conditions investigated. In addition, the ct chain of canine Hp was found to contain carbohydrate and its amino terminus was found to be partially blocked by the occurrence of pyrrolidonecarboxylic acid. Acknowledqements--We wish to thank Mrs Fu-Mei Lo. Mr Horace D. Kelso, Mr Billy Touchstone and Mrs Linda Merryman for valuable technical assistance.
REFERENCES AMBLER R. P. (1972) Carboxypeptidases A and B. Meth. Enzym. 25, 262-272. BANKER G. A. & COTMANC. W. 0972) Measurement of free electrophoretic mobility and retardation coefficient of protein-sodium dodecyl sulfate complexes by gel electrophoresis. J. biol. Chem. 247, 5856-5861. BECKMAN L. & CEDERMARKG. (1960) Haptoglobin types in Maeaca irus. Acta genet. Statist. reed. 10, 23-27. BLACK J, A. & DIXON G. H. (1968) Amino-acid sequence of alpha chains of human haptoglobins. Nature, Lond.
218, 736-741. FRANCOIS C., MARSHALLR. D. & NEUBERGER A. (1962) Carbohydrates in protein. Biochem. J. 83, 335-341. FULLER G. M., RASCO M. A., McCOMBS M. L., BARNETT D. R. & BOWMAN B. H. (1973) Subunit composition of haptoglobin 2-2 polymers. Biochemistry, N.Y. 12,
259258. GANROT K. (1973) Plasma protein response in experimental inflammation in the dog. Res. exp. Med. 161, 251-261. HARVEY J. W. (1976) Quantitative determinations of normal horse, cat and dog haptoglobins. Theriogenology 6, 133-138. KONG S.-K. & SHIM B.-S. (1974) Studies on equine serum haptoglobin: Purification and partial characterization. J. Cath. Med. Coll. (Seoul) 26, 7-19. KURACm K. & DAVIE E. W. (1977) Activation of human factor XI (plasma thromboplastin antecedent) by factor Xlla (activated Hageman factor). Biochemistry, N.Y. 16, 5831-5839. KUROSKYA., BARNETTD. R., RASCO M. A., LEE T.-H. & BOWMAN B. H. (1974) Evidence of homology between the fl-chain of human haptoglobin and the chymotrypsin family of serine proteases. Biochem. Genet. 11, 279-293. KUROSKY A., HAY R. E., KIM H.-H., TOUCHSTONE B., RASCOM. A. & BOWMANB. H. (1976a) Characterization of the cyanogen bromide fragments of the fl chain of human haptoglobin. Biochemistry, N.Y. 15, 5326-5336. KUROSKVA., KIM H.-H. & TOUCHSTONEB. (1976b) Comparative sequence analysis of the N-terminal region of
344
ALEXANDER KUROSKY, REGINE E. HAY and BARBARAH. BOWMAN
rat, rabbit, and dog haptoglobin //-chains. Comp. Biochem. Physiol. 55B, 453~,59. KUROSKY A., MARKEL D. E. & PETERSON J. W. (1977) Covalent structure of the fl chain of cholera enterotoxin. J. biol. Chem. 252, 7257-7264. LEE T.-H., K1M I.-K., HAM J.-K. & SHIM B.-S. (1974) Absence of interchain disulfide bridges in rabbit haptoglobin molecule. Biochem. biophys. Res. Commun. 60, 710-716. LOCKHART W. L., CHUNG W. P. ~1- SMITH n. B. (1972) Studies on the dissociation of porcine haptoglobin. Can. J. Biochem. .50, 775-781. MALCHY B. & DIXON G. H. (1973a) Studies on the interchain disulfides of human haptoglobins. Can. J. Biochem. 51, 249-264. MALCHV B. & DIXON G. H. (1973b) Correction to the amino acid sequence of the ~ chain of human haptoglobin. Can. J. Biochem. 51, 321-322. MALCHY B., RORSTAD O. & DIXON G. H. (1973) The halfmolecule of haptoglobin: studies on the product obtained by the selective cleavage of a haptoglobin disulfide. Can. J. Biochem. 51, 265-273. PASTEWKA J. V., NESS A. T. & PEACOCK A. C. (1975) Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Biochim. biophys. Acta 386, 530-537. PENKE B., FERENCZY R. & KOVACS K. (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal. Biochem. 60, 45-50. PUTNAM F. W. (1975) Haptoglobin. In The Plasma Proreins (Edited by PUTNAM F. W.), Vol. 2, pp. 1-50. Academic Press, New York. SCHWANTES A. R. & TONDO C. V. (1969) Preliminary data on haptoglobins in vertebrates. Rer. Bras. Pesq. Med. Biol. 2, 105-108. SEGREST J. P., JACKSON R. L., ANDREW E. P. & MARCHESI V. T. (1971) Human erythrocyte membrane glycoprotein: a re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis. Biochim. biophys. Res. Commun. 44, 390-395. SHIFRINE M. & STORMONT C. (1973) Hemoglobins, haptoglobins, and transferrins in beagles. Lab. Anita. Sci. 23, 704-706.
SHIM B.-S., YOON C.-S., OH S.-K., LEE T.-H. & KANG Y. S, (1971) Studies on swine and canine serum haptoglobins. Biochem. biophys. Acta 243, 126-136. SHIM B.-S., YOON J. M., K1M U. R. & LEE T.-H. (1975) Structural characterization of polymeric forms of goat and bovine serum haptoglobins: the existence of larger c~-polypeptide chains. Korean ,I. Biochem. 7, 9-15. SMITHIES O., CONNELL G. E. & DIXON G. H. (1962a) Inheritance of haptoglobin subtypes. Am. J. hum. Genet. 14, 14-21. SMITHIES O.~ CONNELL G. E. t~ DIXON G. H. (1962b) Chromosomal rearrangements and the evolution of haptoglobin genes. Nature, Lond. 196, 232-236. SUTTON H. E. (1970) The haptoglobins. Pros. reed. Genet. 7, 163-216. SWANK R. T. & MUNKRES K. D. (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 39, 462m,77. TRAVIS J. C. & SANDERS B. G. (1972) Haptoglobin evolution: Polymeric forms of Hp in the bovidae and cervidae families. J. exp. Zool. 180, 141-148. TRAVIS J. C., GARZA J. & SANDERS B. G. (1975) Structural characterization of polymeric haptoglobin from goats. Comp. Biochem. Physiol. 51B, 93-97. USPENSKAYA V. D., PLESKOVAM. V. ~g NEDELYAEVAM. 1. (1968) Large scale haptoglobin isolation from the its component polypeptide chains. Biokhimiya 32, 347 353. USPENSKAYA V. D., PLESKOVAM. V. & NEDELYAEVA M. I. (1968) Large scale haptoglobin isolation from the blood of dogs. Vop. reed. Khim. 14, 324-329. WARREN L. (1959) The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975. WEBER K. & OSBORN M. (1969)The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. WOODWORTH R. C. • CLARK L. G. (1967) An improved vertical polyacrylamide gel electrophoresis apparatus. Anal. Bioehem. 18, 295-304. ZACHARIAS R. M., ZELL T. E., MORRISON J. H. & WOODLOCK J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148-152.
0305-0491 79'0401-0339502.00'0
CANINE HAPTOGLOBIN" A U N I Q U E HAPTOGLOBIN SUBUNIT ARRANGEMENT* ALEXANDER KUROSKYi', REGINE E. HAY and BARBARA H. BOWMAN Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550, U.S.A. (Received 7 August 1978)
Abstract--1. Isolated canine haptoglobin behaved identically to the Gt2fl2 structure typical of human haptoglobin type i-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptogiobin dissociated into otfl subunits that separated into • and fl chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine ~t chain when compared to human ct~ chain. 4. These results provide evidence that there is no inter ct chain disulphide in canine haptoglobin comparable to the 0tt20~tl20 disulphide in human haptoglobin that links the two ~tfl subunits.
INTRODUCTION
Haptoglobin is a glycoprotein found predominantly in the ~t2 fraction of serum. It is present in sera of all classes of vertebrates studied (Sutton, 1970; Putnam, 1975). The electrophoretic mobility of Hp§ from most animals resembles that of human Hp 1-1 after alkaline gel electrophoresis (Schwantes & Tondo, 1969; Kurosky et al., 1976b). Notable exceptions are some members of the order Artiodactyla (Travis & Sanders, 1972) and some species of monkey (Beckman & Cedermark, 1960) which demonstrate polymeric Hp analogous to human Hp 2-2 and 2--1. Shifrine & Stormont (1973) examined Hp in sera from 200 beagles and found no evidence of polymorphism or polymer formation. The mean concentration of Hp in the serum of healthy dogs is 1.04 mg/ml, similar to that reported for man (0.89 mg/ml)(Harvey, 1976). Like Hp from other species, canine Hp increases in concentration 4- to 10-fold during inflammation (Ganrot, 1973). The purification and partial characterization of canine haptoglobin have been reported (Kurosky et al., 1976b; Uspenskaya et al., 1967, 1968). In all species investigated that possess nonpolymeric Hp, the functional molecule is a tetrachain structure comprised of two ~t and two fl chains that are covalently linked by interchain disulphide bonds (Smithies et al., 1962a; Malchy & Dixon, 1973a; Malchy et al., 1973; Kurosky et al., 1976a; Shim et al., 1971; Lockhart et al., 1972; Shim et al., 1975; Kong & Shim, 1974). A single disulphide bridge attaches the ~t and fl chains to form an 0~fl subunit in human Hp 1-1. Two such subunits are combined by an ct120-ct120 disulphide bond (Malchy & Dixon, 1973a; Kurosky et al., 1976a). The structure of human
Hp 1-1 is diagrammatically represented in Fig. 1. The one exception in interchain disulphide arrangement in animal Hp was described by Lee et al. (1974). In this report evidence was presented that rabbit Hp contained no interchain disulphides. In most species examined carbohydrate is found only on the hp /~ chain (20Yo in human hp fl chain), except for goat, which was reported to have carbohydrate on its :t chain as well (Travis et al., 1975). This paper compares the interchain disulphide arrangement of human, rat, rabbit, and canine Hp and presents evidence for an interchain disulphide arrangement in canine Hp that is unique among haptoglobins studied thus far. MATERIALS AND METHODS
Plasma was obtained from the following animals: (I) rabbit--an outbred strain of New Zealand Whites; (2) rat--an outbred strain of Sprague-Dawley; and (3) dog-greyhounds. Rat and rabbit plasma represented pooled samples but the plasma obtained from two dogs was processed separately, Pth amino acids and all reagents used in the sequencer were "Sequanal Grade" purchased from Pierce Chemical Co. and Beckman Instruments, Inc. Acrylamide and N,N'-methylenebisacrylamide, "Electrophoresis Purity", were products of Bio-Rad Laboratories. Carboxypeptidase A, treated with diisopropylfluorophosphate, was purchased from Worthington Biochemical Corp. Haptoglobin was isolated as previously described (Kurosky et al., 1976b) from plasma of rat, rabbit, and dog conditioned to acute phase inflammation by turpentine injection. All Hp purification steps were promptly carried out at 4 ° to minimize any possible protein alteration, and only freshly drawn plasma was used. Haptoglobin homogeneity was investigated by polyacrylamide gel electrophoresis (Woodward & Clark, 1967) and by SDS polyacrylamide gel electrophoresis (Weber & Osborne, 1969). * This investigation was supported by grant HD 03321 As previously reported (Kurosky et al., 1976b), the ct and from the National Institute of Child Health and Human fl chains of purified Hp were separated by gel filtration following reduction with 2-mercaptoethanol and alkylation Development. with iodoacetamide. Homogeneity of the isolated chains t To whom correspondence should be addressed. § Abbreviations: Hp, haptoglobin (intact); hp, haptoglo- was established by polyacrylamide gel electrophoresis and bin (chains); Hb, hemoglobin; Pth, phenylthiohydantoin; automated sequence analysis. Molecular weight estimates of intact canine Hp and SDS, sodium dodecyl sulphate; EDTA, ethylenediaminereduced and alkylated Hp were conducted essentially by tetracetic acid. 339
340
ALEXANDER KUROSKY, REGINE E. HAY and BARBARA H. BOWMAN
I05 I S
20 34 I I I
NI
r s--s7 Fs-s7 /~CHAIN 148 179,9o 2B z . ~ c
S
68 I 172 83 C or CHAINS
S
1 ] ' ~ - ~ " t 72 85 C 20 34 68 I
NI
S
I 5
N
[ 105
I
rsI~S7 148
F s-s7 245c ~ CHAIN 179 190 219
Fig. 1. Tetrachain arrangement of human Hp 1-1. Numbering of ct chain residues was revised (Kurosky et al., 1976a). Numbering and disulphide arrangement of fl chain residues were established by sequence analysis (Kurosky, Barnett, Lee, Touchstone and Bowman, in preparation). the methods of Weber & Osborne (1969), Swank & Munkres (1971), and Segrest et al. (1971), as we previously described (Kurosky et al., 1976a). After electrophoresis the gels were stained for protein with Coomassie brilliant blue R or for carbohydrate with the periodic acid-Schiff stain (Zacharias et al., 1969). Gels stained for carbohydrate were first washed overnight with 25% isopropanol/10% acetic acid and then with 10% isopropanol/10% acetic acid before staining. A molecular weight estimate of canine Hp was also made by gel filtration on a calibrated column of Sephadex G-100 (2.5 cm x 110 cm) equilibrated with 0.1% NH4HCO3, pH 8.5. Protein hydrolysis of the ct chain of canine Hp was carried out at 107° in 5.7 N HCI for compositional analysis and at 100° and 4 N HCI for hexosamine analysis. Methods of amino acid analysis were recently described (Kurosky et al., 1977). Quantitation of neutral sugars was made by the Winzler orcinol-sulfuric acid procedure (Fran-
cois et al., 1962) and of sialic acid by the thiobarbituric acid assay (Warren, 1959). Reaction with carboxypeptidase A was carried out according to Ambler (1972). Sequencer operation and methods of identification and quantitation of Pth amino acids were as previously detailed (Kurosky et al., 1976b). RESULTS Comparative acrylamide gel electrophoresis of rat, rabbit, dog, and human Hp indicated that the electrophoretic mobility at pH 8.9 was similar for all four species of Hp (Fig. 2a). In the presence of urea the electrophoretic mobilities of rat, rabbit, and human Hp were identical but the mobility of canine Hp was significantly faster (Fig. 2b). The elution volume of canine Hp on Sephadex G-100 corresponded to an !
~ii~ ~ ~i
: ii ¸¸¸
~
//
i
/
(+)
~i,i~ii~l{ (i I I!i ~~I! ~~;
li :::, !i:i:i: ......,. i:ii:
!
i
~
i i~ii~i~!~i:~i¸
~!i~!i~i~i~i~Ii~i~i~i;li~!~!~i i~!i~i~i!~~~i:~i~, ;%~ i,i!,ii!ii~
!]iiI:
i~!~!i{iii~i!i!~i~i!!i!ii~!~i~i;i!~i~i~:~~i:~,~:i~i~ ¸ ~il~i~;~i:~!i~:ii~iil~
1 Hu
'!i: Do
Rt (a)
Origin
C-) Rb
Rb Rt
Do Hu (b)
Fig. 2. Slab polyacrylamide gel electrophoresis of human (Hu), dog (Do), rat (Rt), and rabbit (Rb) Hp. Gel buffer, 0.1 M tris-borate-EDTA-lactate, pH 8.9, 5% acrylamide (Woodworth & Clark, 1969). (a) without urea; (b) contained 6 M urea. 200pg of protein per slot, stained with Coomassie blue.
Canine haptoglobin subunit arrangement
341
Table 1. Comparison of the molecular weight of canine haptoglobin with rat, rabbit and human haptoglobin ' Canine
Rat
Rabbit
Human i-i
Observed
Corrected b
Observed
Corrected b
Observed
Corrected b
Observed
Sequence c
Intact Hpd
48,500
37,100
102,200
78,100
111,200
85,000
113,600
86,850
a Chaine
19,500
ii,i00
16,300
9,300
16,300
9,300
16,100
9,189
Chaine
33,900
29,400
35,300
30,600
38,500
33,300
39,500
34,200
Determined by SDS polyacrylamide gel electrophoresis according to Segrest et al. (1971). b Corrected relative to human Hp 1-1. c From primary structure analysis. d Nonreducing conditions in 5% acrylamide. Hp electrophoresis in 5%, 7.5%, 10%, and 12.5% gels containing 2-mercaptoethanol gave a minimum fl chain molecular weight (observed) at 12.5%. The ~ chain molecular weight was donstant and was averaged.
estimated molecular weight of 100,000 and was the same as human Hp 1-1. The observed molecular weights of the different Hp's and their component chains obtained by gel electrophoresis are summarized in Table 1. After SDS polyacrylamide gel electrophoresis, canine ~ and fl chains and human fl chain stained positively with the periodic acid-Schiff reagent but human ~ chain did not. The amino acid composition of carbamoylmethylated at chain of canine Hp is compared to human ~t chain in Table 2. Hydrolysis of canine ~t chain with carboxypeptidase A is shown in Fig. 3. Automated sequence analysis of the ~ chain of canine H p revealed the amino-terminus to be partially blocked due to cyclization of a glutaminyl residue giving rise to a pyrrolidonecarboxylyl residue. Since cyclization of the terminal glutamine was not complete, we were able to confirm nine of the amino-terminal residues as indicated in Table 3. Carbohydrate analysis of canine
ct chain indicated 3% neutral hexoses and 3% sialic acid. DISCUSSION In nondissociating buffers, canine Hp behaved similarly to human Hp during both polyacrylamide gel electrophoresis and gel filtration. However, in dissociating buffers, either urea or SDS, the protein migrated like an ~fl subunit rather than a n ~2fl2 tetrachain molecule. The slower mobility of rat Hp when compared to human Hp (Fig. 2a) was the result of aggregation as evidenced by the diffuse pattern of staining. In the presence of urea (Fig. 2b) the mobility of rat Hp was more like that of human Hp. In contrast to the report of Lee et al. (1974), rabbit Hp could not be separated into ~ and fl chains without the addition of a reducing agent. Heating rabbit Hp in the presence of I% SDS at 60 ° for two hr also
Table 2. Comparison of the amino acid compositions of canine ct and human :d chains of Hp" Canine a chain b
Lysine Histidine Arginine CM-cysteine d Aspartic acid Threonine d Serine d Glutamic acid Proline Glycine Alanine
7.8±.2 3.0±.I 1.2±.i 3.0±.I 8.6±.3 5.9±.I 2.8±.I 13.6±.3 6.9±.4 5.9±.2 2.8±.i
(8) (3) (i) (3) (8-9) (6) (3) (13-14) (7) (6) (3)
Human a I chain c
Amino Acid
8 2 2 4 14 3 2 9 7 7 5
Valine Methionine d Isoelucine Leucine Tyrosine Phenylalanine Tryptophan e Glucosamine f Galactosamine
Canine a chain b
7.7±.2 0.8±.1 1.9±.2 4.0±.2 3.7±.I 1.0±.03 1.1 4.0 0
Total Amino Acids
(8) (1) (2) (4) (4) (i) (i) (4)
82-84
Human al chain c
8 0 3 3 5 0 i 0 0 83
Values calculated from duplicate time-course hydrolyses: 24, 48, and 96 hr. b Residues per mol of protein based on the molecular weight of human ~1 chain (MW 9, 189). Residues established by sequence analysis (Black & Dixon, 1968; Malchy & Dixon, 1973b; Kurosky et al., 1976a). d Zero time of hydrolysis calculated from linear regression analysis. e Average of duplicate hydrolyses after reaction with mercaptoethanesulfonic acid (Penke et al., 1974). r Time-course hydrolysis for 6, 12, and 18 hr established maximum release to occur at 12 hr.
342
ALEXANDER KUROSKY, REGINE E. HAY a n d BARBARA H. BOWMAN 1
I
I
I
2
3
I
,
4
5
//
,
1.0c-~ la3
co
0.8-
h.I t.d
QO Q6~m b] Ou "~n-"
._1
~
0.2-
3~3
TIME (rain) Fig. 3. Carboxypeptidase A hydrolysis of carbamoylmethylated canine ~ chain at 37" (0) glutamine, (O1 valine; enzyme/substrate = 1/150 (w/w). (I) glutamine, ([]) valine, enzyme/substrate = 1/15 (w/w). Molecular weight of polypeptide portion determined to be 9,314 from compositional analysis (see Table 2j. Table 3. Comparison of the amino-terminal sequence of canine ~ chain with human ~ chain I
2
3
4
5
6
7
8
9
Human a
Val-Asn-Ser-Gly-Asn-Asp-Val-Thr-Asp /
Canine b
Gln-Asn-Thr-Gly-Ser-Gln-Ala-Thr-Ala/
Revised sequence, see Kurosky et al. (1976a). b Established by automated sequence analysis. Partially blocked (80%) by Gin cyclization. did not alter its mobility on SDS polyacrylamide gel electrophoresis. The observed molecular weights of the various animal Hp's and their corresponding chains from SDS polyacrylamide gel electrophoresis were corrected for anomalous mobility as shown in Table 1. These corrections were based on the molecular weight of human Hp calculated from compositional and sequence analysis (Kurosky et al., 1976a). The observed/3 chain values were about 12% exaggerated and intact Hp was elevated by about 22% due to carbohydrate attachment. Malchy et al. (1973) have also reported similar anomalous Hp mobility on SDS polyacrylamide gels. The electrophoretic mobility of human ~1 chain in SDS, as well as in SDS-urea polyacrylamide gels was consistently higher than expected based on the molecular weight determined by sequence analysis (Black & Dixon, 1968; Malchy & Dixon, 1973b; Kurosky et al., 1976a). The exact cause of this anomalous mobility is presently unclear. One possible explanation may be that the relatively high percentage of prolyl residues (8.4%) clustered at positions 17 and 78 restricts free rotation and gives rise to a more extended molecule. The occurrence of chain dimers was unlikely since prior heating of ~1 chain in 1% SDS and 1% 2-mercaptoethanol at 100°
for 10 min followed by 4 hr at 60° did not alter its mobility during SDS gel electrophoresis. The anomalous mobility of the hp al chain appears to be related to the gel electrophoretic system employed since ultracentrifugation studies gave a molecular weight of 8,860 + 400 (Smithies et al., 1962b). Anomalous mobility during polyacrylamide gel electrophoresis has been reported for a number of other proteins (Swank & Munkres, 1971; Banker & Cotman, 1972). The molecular weights of the • chains of rat and rabbit Hp are comparable to that of human (MW 9,189); however, the molecular weight of canine chain is comparatively larger as a result of carbohydrate attachment. Evidence that the molecular weight of the polypeptide portion of canine ~ chain is similar to human ~1 chain is supported by compositional analysis (Table 2) which gave nearly integral values and indicated 82 and 84 residues as compared to 83 residues for human a t chain. In addition, the molar release of carboxyl-terminal residues by carboxypeptidase A (Fig. 3) was in agreement with the polypeptide molecular weight calculated from compositional analysis (MW 9,314). Amino acid compositional analysis of the at chain of canine Hp indicated only three half-cystinyl residues compared to four reported for the cta chain of
Canine haptoglobin subunit arrangement human Hp. This result was consistent with the electrophoretic evidence and suggests that the half-cystinyl residue comparable to ct~20 in human Hp is replaced in canine ~ chain by some other amino acid. Unlike human ct chain, canine ~t chain contains a phenylalanyl and a methionyl residue. In addition, four residues of glucosamine were calculated per mol of canine :t chain which indicated carbohydrate attachment in agreement with the positive reaction obtained with the periodic acid-Schiff reagent. Automated sequence analysis of the amino terminus of canine ~t chain identified nine residues, six of which were different but chemically similar to comparable residues in human ct chain. Although this is only a small segment of ct chain, it is distinctly more variable than similar amino-terminal segments of canine and human fl chains (Kurosky et al., 1976b). Sequencer analysis of intact canine Hp indicated that the ~ chain was about 60~o blocked due to cyclization of its amino-terminal glutaminyl residue. The degree of cyclization increased to about 80% for purified ~t chain as a result of chemical manipulation during the isolation procedure. Hydrolysis of dog hp ct chain with carboxypeptidase A revealed a carboxyl-terminal sequence of valyl-glutamine identical to human ~t chain. However, the residue adjacent to valine must not be proline, as in the human, since carboxypeptidase A does not normally release residues preceded by proline. These results agree with the carboxyl-terminal evidence of dog Hp as reported by Uspenskaya et al. (1967). Investigation of the Hb binding capacity revealed that canine Hp was able to bind Hb as effectively as human Hp indicating that the inter :t chain disulphide linkage in canine Hp is not essential for Hb binding. Although the inter ct disulphide is absent, the ctfl subunit of canine Hp dimerizes to give a tetrachain structure, i.e., (ctfl)2, as judged by its electrophoretic mobility in nondissociating conditions (Fig. la) and by gel filtration on Sephadex G-100. The dimer is readily dissociated in the presence of urea or SDS. Interestingly, selective cleavage of the inter ~ disulphide in human Hp 1-1 by sulphitolysis does not disrupt its tetrachain structure as evidenced by electrophoresis in alkaline borate buffer (Malchy et al., 1973). Therefore, although a tetrachain structure is indicated for most animal haptoglobins investigated to date, it is apparent that other subunit or chain arrangements are also possible as indicated by the (~tfl)2 structure of canine Hp. Moreover, the human polymorphic Hp types 2-1 and 2-2, which give rise to a polymeric series covalently bonded by disulphide linkage (Fuller et al., 1973; Pastewka et al., 1975), are additional examples of variation of Hp subunit structure. All of these structural variations are capable of binding Hb. It is not uncommon that families of homologous proteins demonstrate multiple chain or subunit arrangements, e.g., the serine proteases. In view of the homology of Hp to the chymotrypsinogen family of serine proteases (Kurosky et al., 1974), it is worthwhile to point out that human clotting factor XIa is a tetrachain structure comprised of a pair of heavy chains and a pair of light chains held together by disulphide bonds (Kurachi & Davie, 1977). Haptoglobin from both dogs (greyhounds) used in
343
this study gave identical electrophoretic patterns on polyacrylamide gels. It will be of interest to examine the disulphide arrangement of Hp isolated from other breeds of dogs as well as from related families in order to define the extent of genetic variation of ct chain in the order Carnivora. SUMMARY
Electrophoretic characterization of canine Hp under dissociating conditions revealed a molecular weight corresponding to an ~tfl structure rather than an ct2fl2 chain arrangement identified for most nonpolymeric animal Hp. Compositional analysis of canine :t chain indicated only three half-cystinyl residues which, together with the electrophoretic evidence, suggested that the inter ~t chain disulphide comparable to Cys-20-Cys-20 in human ct~ chain is absent. The ~tfl subunit of canine Hp occurred as a dimer, (~tfl)2, under nondissociating conditions investigated. In addition, the ct chain of canine Hp was found to contain carbohydrate and its amino terminus was found to be partially blocked by the occurrence of pyrrolidonecarboxylic acid. Acknowledqements--We wish to thank Mrs Fu-Mei Lo. Mr Horace D. Kelso, Mr Billy Touchstone and Mrs Linda Merryman for valuable technical assistance.
REFERENCES AMBLER R. P. (1972) Carboxypeptidases A and B. Meth. Enzym. 25, 262-272. BANKER G. A. & COTMANC. W. 0972) Measurement of free electrophoretic mobility and retardation coefficient of protein-sodium dodecyl sulfate complexes by gel electrophoresis. J. biol. Chem. 247, 5856-5861. BECKMAN L. & CEDERMARKG. (1960) Haptoglobin types in Maeaca irus. Acta genet. Statist. reed. 10, 23-27. BLACK J, A. & DIXON G. H. (1968) Amino-acid sequence of alpha chains of human haptoglobins. Nature, Lond.
218, 736-741. FRANCOIS C., MARSHALLR. D. & NEUBERGER A. (1962) Carbohydrates in protein. Biochem. J. 83, 335-341. FULLER G. M., RASCO M. A., McCOMBS M. L., BARNETT D. R. & BOWMAN B. H. (1973) Subunit composition of haptoglobin 2-2 polymers. Biochemistry, N.Y. 12,
259258. GANROT K. (1973) Plasma protein response in experimental inflammation in the dog. Res. exp. Med. 161, 251-261. HARVEY J. W. (1976) Quantitative determinations of normal horse, cat and dog haptoglobins. Theriogenology 6, 133-138. KONG S.-K. & SHIM B.-S. (1974) Studies on equine serum haptoglobin: Purification and partial characterization. J. Cath. Med. Coll. (Seoul) 26, 7-19. KURACm K. & DAVIE E. W. (1977) Activation of human factor XI (plasma thromboplastin antecedent) by factor Xlla (activated Hageman factor). Biochemistry, N.Y. 16, 5831-5839. KUROSKYA., BARNETTD. R., RASCO M. A., LEE T.-H. & BOWMAN B. H. (1974) Evidence of homology between the fl-chain of human haptoglobin and the chymotrypsin family of serine proteases. Biochem. Genet. 11, 279-293. KUROSKY A., HAY R. E., KIM H.-H., TOUCHSTONE B., RASCOM. A. & BOWMANB. H. (1976a) Characterization of the cyanogen bromide fragments of the fl chain of human haptoglobin. Biochemistry, N.Y. 15, 5326-5336. KUROSKVA., KIM H.-H. & TOUCHSTONEB. (1976b) Comparative sequence analysis of the N-terminal region of
344
ALEXANDER KUROSKY, REGINE E. HAY and BARBARAH. BOWMAN
rat, rabbit, and dog haptoglobin //-chains. Comp. Biochem. Physiol. 55B, 453~,59. KUROSKY A., MARKEL D. E. & PETERSON J. W. (1977) Covalent structure of the fl chain of cholera enterotoxin. J. biol. Chem. 252, 7257-7264. LEE T.-H., K1M I.-K., HAM J.-K. & SHIM B.-S. (1974) Absence of interchain disulfide bridges in rabbit haptoglobin molecule. Biochem. biophys. Res. Commun. 60, 710-716. LOCKHART W. L., CHUNG W. P. ~1- SMITH n. B. (1972) Studies on the dissociation of porcine haptoglobin. Can. J. Biochem. .50, 775-781. MALCHY B. & DIXON G. H. (1973a) Studies on the interchain disulfides of human haptoglobins. Can. J. Biochem. 51, 249-264. MALCHV B. & DIXON G. H. (1973b) Correction to the amino acid sequence of the ~ chain of human haptoglobin. Can. J. Biochem. 51, 321-322. MALCHY B., RORSTAD O. & DIXON G. H. (1973) The halfmolecule of haptoglobin: studies on the product obtained by the selective cleavage of a haptoglobin disulfide. Can. J. Biochem. 51, 265-273. PASTEWKA J. V., NESS A. T. & PEACOCK A. C. (1975) Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Biochim. biophys. Acta 386, 530-537. PENKE B., FERENCZY R. & KOVACS K. (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal. Biochem. 60, 45-50. PUTNAM F. W. (1975) Haptoglobin. In The Plasma Proreins (Edited by PUTNAM F. W.), Vol. 2, pp. 1-50. Academic Press, New York. SCHWANTES A. R. & TONDO C. V. (1969) Preliminary data on haptoglobins in vertebrates. Rer. Bras. Pesq. Med. Biol. 2, 105-108. SEGREST J. P., JACKSON R. L., ANDREW E. P. & MARCHESI V. T. (1971) Human erythrocyte membrane glycoprotein: a re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis. Biochim. biophys. Res. Commun. 44, 390-395. SHIFRINE M. & STORMONT C. (1973) Hemoglobins, haptoglobins, and transferrins in beagles. Lab. Anita. Sci. 23, 704-706.
SHIM B.-S., YOON C.-S., OH S.-K., LEE T.-H. & KANG Y. S, (1971) Studies on swine and canine serum haptoglobins. Biochem. biophys. Acta 243, 126-136. SHIM B.-S., YOON J. M., K1M U. R. & LEE T.-H. (1975) Structural characterization of polymeric forms of goat and bovine serum haptoglobins: the existence of larger c~-polypeptide chains. Korean ,I. Biochem. 7, 9-15. SMITHIES O., CONNELL G. E. & DIXON G. H. (1962a) Inheritance of haptoglobin subtypes. Am. J. hum. Genet. 14, 14-21. SMITHIES O.~ CONNELL G. E. t~ DIXON G. H. (1962b) Chromosomal rearrangements and the evolution of haptoglobin genes. Nature, Lond. 196, 232-236. SUTTON H. E. (1970) The haptoglobins. Pros. reed. Genet. 7, 163-216. SWANK R. T. & MUNKRES K. D. (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 39, 462m,77. TRAVIS J. C. & SANDERS B. G. (1972) Haptoglobin evolution: Polymeric forms of Hp in the bovidae and cervidae families. J. exp. Zool. 180, 141-148. TRAVIS J. C., GARZA J. & SANDERS B. G. (1975) Structural characterization of polymeric haptoglobin from goats. Comp. Biochem. Physiol. 51B, 93-97. USPENSKAYA V. D., PLESKOVAM. V. ~g NEDELYAEVAM. 1. (1968) Large scale haptoglobin isolation from the its component polypeptide chains. Biokhimiya 32, 347 353. USPENSKAYA V. D., PLESKOVAM. V. & NEDELYAEVA M. I. (1968) Large scale haptoglobin isolation from the blood of dogs. Vop. reed. Khim. 14, 324-329. WARREN L. (1959) The thiobarbituric acid assay of sialic acids. J. biol. Chem. 234, 1971-1975. WEBER K. & OSBORN M. (1969)The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. WOODWORTH R. C. • CLARK L. G. (1967) An improved vertical polyacrylamide gel electrophoresis apparatus. Anal. Bioehem. 18, 295-304. ZACHARIAS R. M., ZELL T. E., MORRISON J. H. & WOODLOCK J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148-152.
