Comp. Biochem. Physiol., 1976, Vol. 55B, pp. 453 to 459. Pergaraan Press. Printed In Great Britain

COMPARATIVE SEQUENCE ANALYSIS OF THE N-TERMINAL REGION OF RAT, RABBIT,

AND DOG HAPTOGLOBIN fl-CHAINS*

ALEXANDER KUROSKYt, HAN-HWA KLM~/ AND BILLY TOUCHSTONE Division of Human Genetics, Department of Human Biological Chemistry and Genetics,

The University of Texas Medical Branch, Galveston, TX 77550, U.S.A.

(Received 21 January 1976)

Abstract--1. Quantitative sequence analysis of a 40-residue segment of the amino-terminal region of the ~-chain of haptogiobin isolated from sera of rat, rabbit, and dog was undertaken.

2. All three haptoglobin ~-chains aligned precisely with human ~-chain with no evidence of deletions or insertions.

3. The ~ difference in amino acid residues between the haptoglobin fl-chains was comparable to that reported for the fl-chains of hemoglobin from identical mammalian sources.

4. Human and dog haptoglobin fl-chains had carbohydrate attached to asparagine at residue-23 whereas rat and rabbit did not.

INTRODUCTION

Amino acid sequence analysis of the fl-chain of human Hp~ has provided strong evidence of homo- logy between the fl-chain and the chymotrypsin family of serine proteases (Barnett et al., 1970, 1972; Kurosky et al., 1974a, 1974b). In a comparison of some 171 residues of human Hp/i-chain (60% of the molecule) approximately 30~ of these were identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsins A or B, porcine elas- tase, or bovine thrombin B-chain. These sequence similarities give evidence that Hp is an important addition to our present knowledge of the evolution of the serine protease class of enzymes. A number of proteins have now been reported which have modi- fied or different functions but are evolutionarily related, e.g~ growth hormone, prolactin, and placental lactogen CNiall et al., 1971); lactalbumin and lyzo- zyme (Hill et al., 1969). These examples provide evi- dence for the existence of protein superfamilies con- taining proteins of recognizably related sequences (Dayhoff et al., 1975).

In the case of Hp, the preservation of structural similarity to the serine proteases may be related to the nature of the new function of Hp after its diver- gence from the proteases, i.e. binding of Hb. If the binding of Hb is indeed an important function of Hp, then the evolutionary constraints due to the interac- tion of such a large "substrate" as Hb could be signifi- cant. Since the association constant for the reaction

*This investigation was supported by grants from the National Institutes of Health, HD 03321, and the Robert A. Welch Foundation, H-378.

1" To whom correspondence should be addressed. ~:Present address: Department of Biology, Catholic

Medical College, Seoul, Korea. §Abbreviations: Hp, haptoglobin; Hb, hemoglobin;

DEAE, dicthylaminoethyl; PTH, phenylthiohydantoin; DMBA, N,N-dimethylbenzylamine; HI, hydriodic acid; SDS, sodium dodecyl sulphate.

of Hp with Hb indicates a virtually irreversible inter- action (5 x 106 M -a, Chiancone et al., 1968), it is likely that the binding of Hb utilizes an extensive binding region which could involve a considerable portion of the Hp molecule. This view is further sup- ported by the calorimetric determinations of Lavialle et al., (1974), who found that the Hp-Hb association is similar to the refolding reaction of proteins since the entropy changes, the enthalpy, and heat capacity changes are all large and negative. It is known that one molecule of Hp 1-1 binds two =fl subunits of Hb; however, the binding to Hb is attributed primar- ily to the//-chain of Hp rather than the ~-chaln (Gor- don et al., 1966, 1968). In view of the above consider- ations as well as the sequence similarities with the serine proteases, one would expect that the Hp fl-chain, or portions thereof, might demonstrate a conservative rate of mutation. It will be important to define whether the regions of preservation of pri- mary structure are related to the binding site of Hb as suggested by the comparative immunological studies of Javid & Fuhrman, (1971).

In order to further evaluate the evolution of Hp we have undertaken sequence analysis of Hp B-chain isolated from sera of rat, rabbit, and dog. We report here quantitative sequence analyses of the N-terminal region of rat, rabbit, and dog Hp B-chains as deter- mined by automated techniques. These mammalian sequences are compared with previously reported fl-chain sequences of human Hp (Barnett et al., 1972; Kurosky et al., 1974a). A preliminary account of a portion of this work was presented elsewhere (Kur- osky et al., 1974b).

MATERIALS

PTH amino acids and all reagents used in the sequencer were "Scquanal Grade" purchased from Pierce Chemical Co. Iodo [1-~4C'1 acetamide, specific activity 49 mCi/m- mole, phenyl 1~4C] isothiocyanate, specific activity 11.6 mCi/m-mole, were obtained from Amersham/Searle Cor- poration. SP-400 gas chromatographic column-packing material (pre-coated) was a product of Beckman Instru-

453

454 ALEXANDER KUROSKY, HAN-HwA KIM AND BILLY TOUCHSTONE

ments, Inc. N,O-bis (trimethylsilyl) acetamide was pur- chased from Supelco, Inc., Bellefonte, PA. The ultrafilter (UM10) was obtained from Amicon Corporation. Human Hp antisera was prepared by Behring Diagnostics.

METHODS

Haptoylobin purification

Rat, rabbit, and dog Hp preparations were fractionated from serum and plasma by modification of the method described by Connell & Shaw, (1961) for human Hp. The animals were conditioned to acute phase inflammation by injection with turpentine (Lombart et al., 1965; Uspens- kaya et al., 1968). Rabbit Hp was isolated from serum by precipitation with ammonium sulfate at 55% saturation. The precipitate was solubilized with, and extensively dia- lyzed against, 0.03 M sodium acetate buffer, pH 4.7, prior to application onto a DEAE-cellulose column (Whatman Celex D~regenerated before use) equilibrated with the same buffer. The unbound protein was washed from the column with the same pH 4.7 buffer. Hp was eluted from the column with a buffer gradient of 0.03 M sodium ace- tate, pH 4.7 and 0.3 M sodium acetate, pH 5.1. The frac- tions containing Hp were pooled and concentrated at 10-15 mg/ml by ultrafiltration on a UMI0 membrane. Dog and rat Hp were isolated from plasma by precipitation with ammonium sulfate between 25% and 55% saturation. Before DEAE chromatography these Hp precipitates were dialyzed against 0.01 M sodium acetate buffer, pH 4.7. Chromatographic fractions were eluted with a gradient of 0.01 M sodium acetate, pH 4.7 and 0.1 M sodium acetate, pH 5.1. Samples containing 250 mg of Hp fractionated on DEAE-cellulose were finally purified by gel filtration on a column of Sephadex G-200 eluted with 0.05 M tris-HC1, pH 8.2, containing 0.2 M NaC1. The fractions containing Hp were pooled, dialyzed against water, and lyophilized. The presence of Hp was determined by stain- ing with 3,Y-dimethoxybenzidine after gel electrophoresis since immunological detection of either rat or dog Hp by precipitation reaction with anti-human Hp (produced in rabbits) gave negative results.

We have found that better Hp recoveries were obtained if, after ammonium sulfate precipitation, the dialyzed pre- cipitate was reasonably dilute (about 1 mg/ml). Excessive concentration at this step will result in considerable preci- pitation of Hp in the relatively low ionic strength condi- tions of the 0.01 M or 0.03 M acetate buffer.

S-Carbamoylmethylation of reduced haptoglobin The haptoglobin preparations were reduced with 2-mer-

captoethanol and alkylated with iodoacetamide (Hirs, 1967). The ~- and /]-chains were separated on a column of Sephadex G-75 eluted with 0.1 M propionic acid. More recent Hp preparations were alkylated with iodo (1-14C) acetamide to facilitate the characterization of half-cystinyl residues during sequence analysis. Purity of the Hp /]-chains was assessed by SDS polyacrylamide gel electro- phoresis (Weber & Osborn, 1969) and automated sequence analysis.

Automated sequence analysis The /]-chains were subjected to sequence analysis with

an 890B Beckman Sequencer (updated with an undercut cup) using the DMBA program described by Hermod- son et al., (1972), (Beckman DMBA program No. 102473). Ethanethiol was added to the n-chlorobutane as well as to the HC1 and ethyl acetate used for the conversion and extraction of the PTH derivatives of the amino acid resi- dues. Proteins in the reaction cup were conditioned by initiating the sequencer program at the cyclization oper- ation. In the case of the /]-chain of Hp, we found that a better film was obtained if during conditioning the pro-

gram was held at the cleavage step to obtain complete solubilization of the glycoprotein in the heptaflurobutyric acid. n-Chlorobutane extracts of the thiazolinone deriva- tives of the amino acid residues were not automatically dried in the sequencer but were dried at about 50 ° under a stream of purified nitrogen. Each collection tube con- tained exactly 50 nmoles of PTH-norleucine as internal standard. The ethyl acetate and aqueous phases were sep- arately analyzed for PTH amino acid residues.

Identification of sequencer products Identification of the PTH derivatives after silylation with

N,O-bis (trimethylsilyl) acetamide was achieved by gas chromatography using a Barber-Colman Model 5000 gas chromatograph similar to the procedure of Pisano et al., (1972). Standard solutions of silylated PTH amino acids were injected onto the column for quantitation of the PTH residues produced by the sequencer. Sequencer residues were also identified by amino acid analysis after back- hydrolysis of the PTH derivatives with either hydriodic acid or sodium hydroxide according to Smithies et al., (1971). Alkaline hydrolysis was carried out primarily to confirm the presence of methionine which is destroyed by HI hydrolysis. Quantitation of the sequencer residue prod- ucts after HI hydrolysis was based on HxW standard values obtained from hydrolyzates of standard mixtures of PTH amino acids. Routinely 1/10 of the unknown ethyl acetate extract of the PTH residue was subjected to gas chromatographic analysis and the remainder was hydro- lyzed with HI for amino acid analysis. Aqueous extracts were separately acid hydrolyzed prior to amino acid analy- sis. Except for threonine, arginine, histidine, and lysine each PTH derivative was identified and quantitated by gas chromatography as well as amino acid analysis. The basic amino acids which are found only in the aqueous layer under the given conditions were similarly confirmed by amino acid analysis. No corrections were made for any loss of thiazolinone during the n-chlorobutane extraction from the reaction cup.

RESULTS

Purification of Hp is illustrated in Figs. 1-3. The chromatographic patterns observed for the purifica- tion of Hp from all 3 species were essentially identical to patterns obtained with human Hp. Comparative acrylamide gel electrophoresis indicated that the mobility of dog and rabbit Hp was similar to that of human Hp under the given conditions. Rat Hp differed from human Hp by the slower anodal migra- tion of the H p - H b complex. The electrophoretic pat- terns are shown in Fig. 4. Under the same gel electro- phoretic conditions, human Hp of 2-1 and 2-2 pheno- types can be distinguished by a characteristic pattern of bands of increasing mol. wt which correspond to a polymeric series. No indication of polymerization was observed after gel electrophoresis of dog, rat, or rabbit Hp. This finding is in agreement with reports of others (Lombart et al., 1965; Uspenskaya et al., 1968). SDS polyacrylamide gel electrophoresis of iso- lated Hp fl-ehains revealed only a single stained band. In addition, sequence analyses of the fl-chains revealed only one polypeptide chain for each of the animal species.

Quantitative recovery curves obtained from auto- mated sequence analyses of the three Hp fl-chains appear in Fig. 5. Repetitive yields ranged 93-95% for all analyses. Representative gas chromatograms of selected fl-chain PTH residues are shown in Fig. 6. Gas chromatographic analysis of PTH-tryptophan

Rat, rabbit, and dog Hp fl-chain N-termiaal sequences 455

. . . . . . . . . . o.I

1.6. / ' / 1"

i / / / / I I o 1.2- JL

< N 0.8. ~

o 0.4" /_ Hp

o d.5 r.o ~.'s £o £s o.o~

VOLUME ILl

Fig. 1. DEAE chromatography at 4 ° of dog IIp obtained from 21 of plasma after ammonium sulfate precipitation (between 25 and 55% saturation). Celex D bed vol, 5 cm x 25 cm; flow rate, 25 ml/hr. Gradient 500 ml 0.01 M sodium acetate buffer, plI 4.7 and 500 ml 0.1 M sodium acetate buffer,

pH 5.1. Region indicated by arrow pooled for subsequent purification.

usually gave 3 peaks whereas the unsilylated deriva- tive eluted as a single peak and was more readily identified (Fig. 6). For confirmation of tryptophan at position 35 the entire moun t s of recovered PTH resi- dues at positions 34, 35, and 36 from separate sequence analyses were injected into the gas chroma- tograph unsilylated.

Sequence analysis of dog fl-chain gave negative results at residue 23 with the normally employed identification procedures of gas chromatography and amino acid analysis after HI and alkaline hydrolysis. Evidence obtained after reaction of residue 23 with phenyl(!~'C)isothiocyanate indicated that this position was occupied by asparagine to which carbohydrate was attached. Residue 23 did not extract from the reaction cup with n-chlorobutane because of the hydrophilic character of the carbohydrate moiety. In

2D"

1.0.

.80-

o

.60-

.40-

.20-

° Hp L

. . . . i i . . . . . 0.8 0.9 I 0 I I 12. 13 1.4 I.,.~ 1.6

VOLUME (L)

Fig. 2. Gel filtration at 4 ° of approx 250 nag of the DEAE fraction of dog Hp on Sephadex G-200. Bed vol, 5 cmx 105 cm; buffer, 0.05 M tris-HC1 in 0.2 M NaCI, pH 8.2; up-flow rate, 25 nil/hr. Hp pooled as indicated

by arrow.

one experiment, after 23 degradations, the contents of the sequencer reaction cup were removed, dried, and reacted with 2 N HC! for 4 hr to hydrolyze the carbohydrate portion from the PTH-Asn. Primarily (14C) PTH-Asp was identified after thin layer chroma- tography using the method of Summer et aL, (1973). In a second experiment, the contents of the reaction cup, after 23 degradations, were subjected to chroma- tography on Sephadex G-25 and Aminex A5 (Bio-Rad Laboratories). The (t4C)-labeled fraction obtained by gel filtration was deproteinized by ion exchange (Aminex A5). The eluant was collected, lyophilized and hydrolyzed with HI. Amino acid analysis revealed the presence of aspartic acid. Human fl-ehain also has carbohydrate at position 23 (Kurosky et al., 1976). This was confirmed by sequence analysis of

2.0 ¸

1.0

.80

,~ .6o

. 4 0 . ~

.20

,:o ,11 1.2 1;3 1'4 lls ,.6 ~:~

VOLUME(L)

Fig. 3. Gel filtration at 4 ° of 250 mg of the G-200 fraction of dog Hp on Sephadex (3-75 after reduction with 2-mer- captoethanol and alkylation with iodoacetamide. Bed vol, 5 c m x 130 cm; buffer, 0.1 M prop ion ic acid; down-flow

rate, 25 ml/hr.

456 ALEXANDER KUROSKY, HAN-HWA KIM AND BILLY TOUCHSTONE

(~)

- -Hb - - H p - H b

In Fig. 7 the N-terminal amino acid sequences of Hp fl-chains of rat, rabbit, and dog are compared with that reported for human fl-chain (Barnett et al., 1972; Kurosky et al., 1974a). In order to evaluate the homology of the 4 species of Hp fl-chain N-termini, ~o differences were calculated as indicated in Table 1. The non-identities observed between Hp sequences of dog & man, and dog & rabbit are 10~o and 13~o, respectively. The 40 N-terminal residues of rat Hp fl-chain indicate less homology to the fl-chain of dog & man (20%) than to rabbit (13%). These differences reflect a rate of mutation for the N-terminal region of the Hp fl-chain which is comparable to that observed for the Hb family of fl-chains (Dayhoff, 1972).

! i ¸ ii ii i !ii iiii~i~ i ~ !~ i~ i~ i~ ?i~i ~ ! ̧

(-) . . . . . . . . . . Origin

(a) (b) ( c ) ( d ) Fig. 4. Slab acrylamide gel electrophoresis of (a) human, (b) dog, (c) rat, and (d) rabbit Hp complexed with human Hb. Gel buffer, 0.1 M tris-borate-EDTA-lactate, pH 9.1,

5~o acrylamide (Woodworth & Clark, 1967).

an isolated carbohydrate containing tryptic peptide (residues 18-41) in addition to whole protein analysis. Sequence analysis of intact human Hp fl-chain yielded negative results at position 23 after routine residue identification. Strikingly, however, analyses of rat and rabbit fl-chain gave no evidence of a carbohydrate moiety at residue 23, nor elsewhere in the 40 residue segment.

DISCUSSION

Of the 40 N-terminal residues of each species com- pared in Fig. 7, 30 are identical in all 4 species. Only 3 positions (5, 23, & 25) necessitate mutations involv- ing more than a single nucleotide base alteration. It should be noted that the sequence reiteration of resi- dues 2-9 at positions 20--27 reported previously by Barnett et al., (1972), and subsequently compiled by Kurosky et al., (1974a, b), for human fl-chain has been corrected (Kurosky et al., 1976) as indicated in Fig. 7.

Of 7 types of carbohydrate-peptide linkages so far characterized, the glucosaminylasparagine linkage has been most extensively studied. N-acetylglucosamine transferase acts primarily on those polypeptide sequences containing Asn-X-Ser or Asn-X-Thr (Neu- berger et al., 1972). In the case of the fl-chain of rab- bit, residue 25 is valine and the criteria of specificity for the transferase are not satisfied; therefore, carbo- hydrate attachment at residue 23 cannot occur. The absence of carbohydrate in the rat//-chain at position 23 is more obvious--asparagine is replaced by gly- cine. Although some controversy remains concerning the recipient amino acid residue in the biosynthetic glycosylation reaction, i.e. whether it is asparagine or aspartic acid (Neuberger et al., 1972), sequence data

o o z

10c z

8c

LIJ

o o w

W

,c io " 4 0

o

• • o - o

2 0 o o

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A. B.

• o

-o.\ ,

0 ,b • ~b 4b DEGRADATION CYCLE

C.

r °OoOo

o •

oo

5b

Fig. 5. Repetitive yield obtained during automated degradation of 8 mg of Hp fl-chain. (a) dog; (b) rat; and (c) rabbit. O, amino acid analysis; Q, gas chromatographic analysis.

Rat, rabbit, and dog tip ~-chain N-terminal sequences 457

r f

z~

M1 . J

" o

, j _ ~ .

W - I

u~

- I

>-

IR

+ j i

.q - )

= N

. J , j U ~" , >,

h t Ul ..J t t l

--I "J

ed ~" ¢ j t2

- j /

Fig. 6. Selected gas chromatograms of silylated PTH sequenator products from dog. •-chain. Vertical axis represents the detector response (arbitrary units) which was constant for all above analyses. As indicated~by the norleucine peak ($TD), later analyses contained a greater proportion of the sample.

The response of the norleucine peak shown in Cycle 1 represents 10 nmoles,

458 ALEXANDER KUROSKY, HAN-HWA KtM AND BILLY TOUCHSTONE

Hp B Chain

Human

Rat

Rabbit

Dog

Base Changes

1 2 3 4 5 6 7 8 9 I0 II 12 13 14 15

• Ile~Gly G l y ~ A s p Ala Lys Gly Ser Phe Pro Trp Gln

l l • lle Ile Gly Gly Set. Asp Ala Lys G1y Ser Phe Pro Trp Gln

• I1e Ile Gly Gly Ser Leu Asp Ala Lys Gly Ser Phe Pro Trp Gin

• lle~Gly Gly Ser~Asp Ala Lys Gly Set Phe Pro Trp Gln

0 1 0 0 2 1 0 0 0 0 0 0 0 0 0

Hp B Chain

Human

Rat

Rabbit

Dog

Base.Changes

16 17 18 19 2 0 21 22 23 24 25 26 2 7 28 2 9 30 CHO

Ala Lys Met Val S e r ~ H i s A~n Leu Thr Thr Gly Ala Thr Leu

Ala Lys Met ] I le ]Ser lArg lHis ]Gly]Leu Thr Thr Gly Ala Thr Leu

Ala Lys Met - - I I V a l Ser]ArglHls Ash Leu ~h~._~.=.Thr Gly Ala Thr Leu I I

Ala Lys Met Val Ser~HisIHis Asn Leu Thr]Ser[Gly Ala Thr Leu

0 0 0 1 0 1 0 2 0 2 1 0 0 0 0

Hp B Chain 31 32 33 34 35 36 37 38 39 40

Human I l e ~ G l n Trp Leu Leu Thr Thr Ala

Rat I l e l S e r ~ s p ~ G l n Trp Leu Leu Thr Thr Ala I U--'---

Rabbit I l e ]Se r IGlu Gln Trp Leu Leu Thr Thr Ala I I

Dog I le]Asn]Glu Gln Trp Leu Leu Thr Thr Ala

Base Changes 0 1 1 0 0 0 0 0 0 0

Fig. 7. Comparison of N-terminal regions of human, rat, rabbit, and dog Hp fl~hmns. Carbohydra~ attachment indmated by CHO. These results were obtained from 5 separMe sequence analys~ ~ r

rat fl-chain, 6 ~r rabbit fl-chain and 7 ~r dog fl-chain. Residue differences are blocked in.

Table 1. Comparison of ~o difference between the fl-chains of Hp*

Human Dog Rabbit Rat

Human 0 10 13 20 Dog 10 0 15 20 Rabbit 13 15 0 13 Rat 20 20 13 0

* Comparison of 40 N-terminal residues.

indirectly supports glycosylation of asparagine. Rab- bit Hb fl-chain is an example of such indirect evi- dence. The occurrence of asparagine at position 23 without carbohydrate attachment due to the absence of theonine or serine at position 25 strongly suggests that glycosylation takes place on asparagine. Similar sequence evidence is provided by the ribonuclease family of proteins (Beintema et al., 1973). It is interest- ing that in presently available families of homologous proteins containing carbohydrate, absence of carbo- hydrate attachment at a specific locus is generally the result of mutation of asparagine rather than the speci- ficity residues threonine or serine. This probably re- flects the greater mutational flexibility provided by 10 codons for serine and threonine as opposed to only 2 for asparagine. However, more sequence infor- mation of glycoproteins is needed to further substan- tiate this observation.

SUMMARY

Sequence analyses of a 40 residue segment of the amino-terminal region of the fl-chain of Hp isolated

from sera of rat, rabbit, and dog are reported. The degree of homology between the fl-chains of Hp was comparable to the homology reported for the fl-chains of Hb for the same mammalian species. A notable difference in the fl-chains of Hp investigated was observed at position 23. Man and dog fl-chains had a carbohydrate moiety attached at this position whereas rat and rabbit fl-chains did not. It is sug- gested that the conservative rate of evolution observed for the 40 residues of the N-terminal region of Hp (about 159/o of the protein molecule) may be related to the function of binding Hb. It is conceiv- able that in binding a large molecule such as Hb a considerable portion of the Hp molecule participates in the interaction giving rise to considerable evolu- tionary constraints. This view is consistent with the physico-chemical evidence available concerning the H p - H b interaction.

Acknowledgements--We are indebted to Mr. Horace D. Kelso and Mr. Terry M. Ward for expert technical assist- ance. Discussions with Dr. D. R. Barnett and Dr. Regine E. Hay were extremely valuable. We especially thank Dr. Barbara H. Bowman for her encouragement and support of these investigations.

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¢.a.P.(n) 55/3n---1