Chicken α2-proteinase inhibitor: A serum protein homologous with ovoinhibitor of egg white

Biochimica et Biophysica Acta, 371 (1974) 52-62 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 36838 C H I C K E N a2-PROTEINASE INHIBITOR: A SERUM LOGOUS W I T H OVOINHIBITOR OF E G G W H I T E

PROTEIN

HOMO-

ALAN J. BARRETT Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN ( U.K.) (Received June 10th, 1974)

SUMMARY 1. Ovoinhibitor was purified from chicken egg white and used to immunize rabbits. It was shown that sera from chickens of both sexes contained an antigen immunologically identical with ovoinhibitor. The average concentration of the inhibitor in serum was similar to that of ovoinhibitor in egg white, i.e. 1 mg/ml. 2. The serum antigen had a2-mobility in immunoelectropboresis, whereas ovoinhibitor run in the fl-zone, at pH 8.3. Treatment of the two proteins with neuraminidase reduced the mobility of the serum protein, without affecting that of ovoinhibitor, so that the final mobilities of the proteins were similar. 3. Affinity chromatography with insolubilized bovine trypsin or chymotrypsin led to the isolation of the a2-proteinase inhibitor in a highly purified form, in a single purification step. a2-Proteinase inhibitor ran slightly further in polyacrylamide gel gradient electrophoresis than did ovoinhibitor, but both run between serum albumin and transferrin. 4. Purified az-proteinase inhibitor was shown to inhibit bovine trypsin, bovine chymotrypsin, and the major Aspergillus proteinase.

INTRODUCTION Ovoinhibitor is a glycoprotein of about 50 000 molecular weight that was discovered and purified from egg white by Matsushima [1 ]. The protein is responsible for all the inhibition of chymotrypsin and Aspergillus proteinase by egg white, and it has been shown that each molecule has independent binding sites for trypsin, chymotrypsin [2] and elastase. The elastase binding site(s) is probably also responsible for the inhibition of the serine proteinase of Aspergillus species [3]. It is shown in the present paper that chicken serum contains an inhibitor immunologically identical with ovoinhibitor, and therefore presumably homologous with it. The name a2-proteinase inhibitor is proposed for this protein. Few examples of the occurrence of egg white proteins in bird serum have so far been described, but the close structural relationship between ovotransferrin and serum transferrin [4, 5] is noteworthy. a2-Proteinase inhibitor has a wide specificity for serine proteinases, and occurs in sufficient quantity in chicken serum to suggest that it could be of physiological importance.

53 MATERIALS AND METHODS Egg white was obtained from fertile but unincubated, or infertile eggs of two hybrid strains, Sykes Hybrid No. 3 and Rhode Island Red x Light Sussex. Chicken serum was from four hens and two cockerels, all mature birds of the above strains. Rabbit polyvalent antisera against chicken serum proteins were purchased from Mercia Diagnostics Ltd, Watford, Herts, U.K. (product of Dakopatts A/S, Copenhagen, Denmark) and from Wellcome Reagents Ltd, Beckenham BR3 3BS, Kent, U.K. Trypsin (bovine, two times crystallized, from Sigma Chemical Co. Ltd, Kingston-upon-Thames, Surrey KT2 7BH, U.K.) and a-chymotrypsin (bovine, type A4 from Boehringer, London, Corp. Ltd, London W5 2TZ, U.K.) were coupled to Sepharose 4B (Pharmacia, Great Britain, Ltd, London W5 5SS, U.K.) activated with CNBr as described by Porath et al. [6]. Thus, highly activated Sepharose was stirred overnight at 4 °C with a solution of trypsin or chymotrypsin (5 mg/g gel) in 0.1 M NaHCO3 adjusted to pH 9.3 with 1 M NaOH. No direct determination of the amount of enzyme remaining bound after exhaustive washing of the gel was attempted. Trypsin was assayed with a-N-benzoyl-o,L-arginine p-nitroanilide as substrate by a method based on that of Erlanger et al. [7]. The activity of the commercial preparation of trypsin was determined by active site titration with p-nitrophenyl-p'guanidinobenzoate (Cyclo Chemical, Los Angeles, U.S.A.) as described by Chase and Shaw [8]; the material was found to be 61 ~o active, and quantities of trypsin mentioned in the results section have been corrected on this bas;s. Aspergillus oryzae proteinase (Type II) was a crude preparation from Sigma, and it was assayed with azo-casein [9] as substrate. Reaction mixtures (1.0 ml) containing 2 ~ (w/v) azo-casein, and 100 mM Tris-HCl buffer, pH 8.0, were incubated for 60 rain at 37 °C. The reaction was stopped by the addition of 5 ml of 3 ~ (w/v) trichloroacetic acid, and the mixtures were filtered. The E 3 6 6 n m of the trichloroacetic acid-soluble reaction products was determined. Neuraminidase (from Vibrio comma) was purchased from Behringwerke AG, Marburg, Lahn, Germany, in a solution of 500 units/ml. Crystallized bovine serum albumin, and human transferrin, were obtained from Sigma, and carboxymethylcellulose was Whatman CM-52. Chicken ovotransferrin was the generous gift of Dr J. Williams (Department of Biochemistry, University of Bristol) and lysozyme (Grade 1) was purchased from Sigma.

Purification of ovoinhibitor The procedure of Matsushima [1] was used to isolate ovoinhibitor from 4.3 1 of egg white. The material obtained in this way was shown by electrophoresis in the presence of sodium dodecylsulphate (see below) to be contaminated with lysozyme, however. The solution was therefore dialysed against 50 mM potassium phosphate buffer in 100 mM NaC1, pH 6.9, and passed through a column (34 cm3 bed vol.) of CM-cellulose in the same buffer. The final product was then precipitated with acetone and dried in a desiccator at room temperature.

Immunization of rabbits Two New Zealand white rabbits were injected intramuscularly on days 0 and

54 14 with 1 or 5 mg of ovoinhibitor that had been dissolved in 1 ml of 1 ~ NaC1 and emulsified with an equal volume of Freund's complete adjuvant. The animals were exsanguinated after 21-25 days. The sera removed after clotting of the blood were made 0.1 ~o NAN3, and stored at 4 °C.

Immunodiffusion, immunoelectrophoresis and radial immunodiffusion Double immunodiffusion plates (8 cm × 8 cm) were coated with 1 ~ agarose (Sigma) in sodium and potassium phosphate buffer (20 mM, pH 7.0) containing 0.15 M NaCI, and the wells had a capacity of 20/~1. For immunoelectrophoresis, the plates were coated with 1.0~ agarose in 90 mM Tris, 80 mM boric acid, 3 mM disodium EDTA, pH 8.3. The capacity of the antigen wells was 5 #1 and of the trGughs 100 #1; electrophoresis was for 1.5 h at a power of 3 W/plate. After development for 24 h, immuno-plates were soaked in 1% NaCI for 48 h, washed and dried; they were then stained with Coomassie Brilliant blue R(0.1%) in sodium formate-formic acid-ethanol-water (4:7:66:126, w/v/v/v) for 30 min and destained thoroughly in the same solvent. Radial immunodiffusion [10] was in phosphate-buffered 1% agarose gels, of thickness 1.5 ram, containing 1.0% (v/v) of rabbit anti-(ovoinhibitor) serum. The wells were of 5 #1 capacity, and plates were developed for 48 h at 20 °C before being washed and stained. Two measurements of diameter (d) were made for each diffusion ring, and d 2 was related to antigen concentration by use of a linear calibration curve (correlation coefficient 0.99) for purified ovoinhibitor, run on the same plate.

Electrophoresis Polyacrylamide gradient electrophoresis was in gels obtained from Pharmacia, run in an apparatus (GE-4) from the same supplier. The buffer was 90 mM Tris, 80 mM boric acid and 3 mM disodium EDTA, pH 8.3, and the run was continued for 1 6 h a t 125V. Electrophoresis was also carried out in cylindrical polyacrylamide gels (11 ~ T, 0 . 9 ~ C in the terminology of ref. 11). A Tris-glycine-HC1 buffer system (ref. 12, System A), and a Tris-borate-sulphate buffer system containing sodium dodecylsulphate [13] were used. All electrophoresis gels were stained for 1 h in 0.1 ~ Coomassie brilliant blue R in methanol-acetic acid-water (5:2:3, by vol.) and destained in a 2:l:17 mixture of the same solvents, all at 55 °C. The gels were stored in 5 ~o (v/v) acetic acid. In all of the figures, the direction of electrophoresis is downwards, towards the anode. Standard samples of ovotransferrin, ovalbumin and lysozyme were run separately as markers for the identification of the major egg white proteins. RESULTS

Purification of active ovoinhibitor It was convenient to monitor the progress of purification of ovoinhibitor by electrophoresis both with and without sodium dodecylsulphate, since ovoinhibitor ran close to ovotransferrin in the former system and ovalbumin in the latter. Lysozyme, the most persistent contaminant, ran into the gels only in the sodium dodecylsulphate system, but ovoinhibitor always ran as a diffuse zone in the sodium dodecyl-

55 sulphate gels, unlike the majority o f proteins. Lysozyme was removed from the ovoinhibitor preparation by adsorption on to carboxymethyl-cellulose. In agreement with previous reports (e.g. ref. 2) ovoinhibitor was resolved into a group o f bands in the Tris-glycine electrophoresis system, and four bands were resolved still more clearly in gradient gel electrophoresis (Fig. lb). The yield of electrophoretically pure ovoinhibitor (from 4.3 1 of egg white) was 1.6 g. N o determination was made of the concentration of ovoinhibitor in the starting material, but on the basis of an average value of 1.03 g/1 for egg white (see below) the yield was 36 ~o.

""

-O

I

II

III

a

IV

I

II

III

b

Fig. 1. Gel electrophoresis of ovoinhibitor. (a) Electrophoresis in the presence of sodium dodecylsulphate to show the progress of purification of ovoinhibitor: (i) egg white, (ii) after precipitation with (NH,)~SO4 and trichloroacetic acid, (iii) at the end of the Matsushima [1] procedure, and (iv) after treatment with carboxymethyl-cellulose. 10/~g of protein was applied to each gel. The origins are marked (O), and the major protein bands are identified as ovotransferrin (OT), ovoinhibitor (OI), ovalbumin (OA) and lysozyme (L). (b) Polyacrylamide gel gradient electrophoresis of (i) bovine serum albumin (5 pg), (ii) ovoinhibitor (10/~g), and (iii) the same as (ii) with 20 #g of chymotrypsin. The serum albumin lane shows traces of ovoinhibitor overlapping from an adjacent lane. In order to establish that all of the forms of ovoinhibition were functionally active, samples o f ovoinhibitor were mixed with an excess o f chymotrypsin (in the weight ratio 1:2), and run in gel gradient electrophoresis as usual. Each o f the ovoinhibitor bands disappeared almost completely from its usual position, to be replaced by a diffuse zone o f lower mobility in the gel (taken to be an incompletely resolved mixture o f ovoinhibitor--chymotrypsin complexes) (Fig. lb).

56

Properties of antisera ra&ed against ovoinhibitor Sera from the two rabbits injected with ovoinhibitor reacted strongly with ovoinhibitor in immunodiffusion; the serum raised by injection of the larger amounts of immunogen also reacted weakly with several other egg white proteins, but the second showed a high degree of specificity (Fig. 2). The more specific antiserum was used, except as indicated, in the experiments described below.

Fig. 2. The reaction of identity in double immunodiffusion between ovoinhibitor and an antigen present in chicken serum. The wells contained (a) egg white (2 #1), (b) chicken serum (5/~1), (c) purified ovoinhibitor (2.5/~g) and (d) antiserum against ovoinhibitor (20/~1).

Reaction of anti-(ovoinhibitor) serum with chicken serum The antiserum raised against ovoinhibitor was run in double immunodiffusion against ovoinhibitor, egg white and chicken serum (Fig. 2). A reaction of complete immunological identity was obtained between ovoinhibitor, either purified or in whole egg white, and an antigen in chicken serum. Ovoinhibitor, egg white and serum from several chickens were also run in immunoelectrophoresis, the plate being developed with anti-(ovoinhibitor) serum (Fig. 3). Ovoinhibitor, either in fresh egg white or purified, had a low anodal (fl) electrophoretic mobility, whereas the mobility of the serum antigen was greater (a2). Ovoinhibitor and chicken serum were also run in polyacrylamide gel gradient electrophoresis, and the gel slab was cut into strips, some of which were stained, while others were embedded in agarose gel containing anti-(ovoinhibitor) serum. Transverse electrophoresis then produced precipitin "peaks" indicating the position of the

57

Fig. 3. Immunoelectrophoresis of purified ovoinhibitor, egg white and chicken sera, developed with anti-(ovoinhibitor) serum. The wells contained (i) egg white (2 #1), (ii) ovoinhibitor (1/~g), (iii) purified a2-proteinase inhibitor (1.5/~g), (iv) hen serum (2/zl) and (v) cockerel serum (2 #1). All of the troughs contained anti-(ovoinhibitor) serum.

antigens in the acrylamide gel. Ovoinhibitor and the serum antigen had closely similar mobilities in this system, running slightly behind serum albumin. The two commercial antisera against whole chicken serum failed to give any reaction with ovoinhibitor in immunodiffusion, and when the chicken sera were run in immunoelectrophoresis a strong new precipitin arc could be produced by supplementing the commercial antisera with the anti-(ovoinhibitor) serum (Fig. 4).

Assay by radial immunodiffusion of ovoinhibitor and az-proteinase inhibitor Samples (0.1 or 0.2 ~1) of sera and egg white were run on radial immunodiffusion plates simultaneously with standard samples (0.05--0.25 #g) of purified ovoinhibitor. Values obtained for the concentration of a2-proteinase inhibitor and ovoinhibitor (in mg/ml) were: hen serum: 1.02, 0.84, 0.86, 1.06; cockerel serum: 1.45, 1.07 (mean of sera 1.05 ± 0.21 S.D.); egg white: 1.52, 1.28, 0.56, 0.61, 1.56, 0.63 (mean 1.03 4- 0.35 S.D.). Thus, it can be said that serum and egg white each contain about 1 mg/ml of a2-proteinase inhibitor or ovoinhibitor, respectively, (when ovoinhibitor is used as standard) but that individual values are moderately variable.

Purification of a2-proteinase inhibitor by affinity chromatography Feinstein [14] has shown that ovoinhibitor can be purified by affinity chromatography on a Sepharose-chymotrypsin adsorbent. It was reasoned that if the

58

i ii iii iv a

b

v vi :

Fig. 4. Affinity chromatography of chicken serum on insolubilized trypsin. (a) Immunoelectrophoresis. The samples were (i, iv) normal chicken serum (2/d), (it) Peak 1 (2 #1) from the Sepharosetrypsin column and (iii) Peak 2, eluted from the adsorbent at pH 3 (containing 1.5 #g of serum ovoinhibitor). The antiserum troughs contained commercial anti-(chicken serum) serum (67 #1) with 33/zl of (A) phosphate-buffered saline, or (B) anti-(ovoinhibitor) serum. The precipitin arc (arrowed) that appears only in diffusion against anti-(ovoinhibitor) serum was selectivelydeleted from the serum by passage through Sepharose-trypsin, and the antigen was subsequently eluted in a highly purified form. (b) Gel gradient electrophoresis. The samples were (i, vi) commercial bovine serum albumin and human transferrin (5/~g each), (it) ovoinhibitor (t0/~g), (iii) chicken serum (2 btl), (iv) Pool 1 from the Sepharose-trypsin column (2/d), and (v) Pool 2 from the column (containing 3.75/~g of ~2-proteinase inhibitor. The locations of the origin (O), a2-macroglobulin (a2M), transferrin (TF) and serum albumin (SA) are marked. antigen of chicken serum also was an inhibitor of trypsin and chymotrypsin it, too, should be bound by these enzymes on an insoluble support. A column (7 cm 3 bed vol.) of Sepharose 4B with trypsin attached (see Materials and Methods) was prepared, and 7 ml of chicken serum was passed through, followed by 50 ml of 0.50 M NaCI containing 20 m M potassium phosphate buffer, p H 7.3. Fractions comprising the unadsorbed peak of protein were combined. The column was then washed with 0.10 M sodium formate buffer, p H 3.0, containing 0.50 M NaCI. A very small peak of protein was eluted, and the appropriate fractions were combined, dialysed against 0.10 M NaC1 containing 20 m M potassium phosphate buffer, p H 7.0, and concentrated by further dialysis against a concentrated solution of Aquacide (Calbiochem Ltd, London W1H 1AS, U.K.). Samples of the original serum and the two peaks of protein from the column were run in immunoelectrophoresis, the antiserum being anti-(chicken serum proteins) alone, or supplemented with anti-(ovoinhibitor) serum (Fig. 4a). It was found that the ovoinhibitor-like antigen of the normal serum, az-proteinase inhibitor, had been specifically removed by passage through the affinity column. The material eluted from the column at pH 3 gave virtually no reaction with the commercial anti-(chicken serum) serum, but contained the %-proteinase inhibitor.

59 The two protein pools from Sepharose-trypsin were also run in polyacrylamide gradient gel electrophoresis (Fig. 4b). Peak one contained the bulk of the serum proteins, with some changes of mobility that might be attributed to proteolysis during passage through the column. Peak 2 contained material running slightly behind albumin (i.e. in the position of the ovoinhibitor-like antigen with only the faintest traces of one other protein, which had the mobility characteristic of a2-macroglobulin. Similar results were obtained when Sepharose-chymotrypsin was used as adsorbent. The yield of aE-proteinase inhibitor from 7 ml of serum run on the Sepharosetrypsin column was 1.0 rag, as determined by radial immunodiffusion with ovoinhibitor as standard. Since all of the serum ovoinhibitor was removed from the serum, the bulk of it evidently remained on the adsorbent, perhaps with other proteinase inhibitors of the serum that might otherwise have contaminated the eluted material. Enough a2-proteinase inhibitor was purified in this way, however, for further experiments.

Effect of neuraminidase on the electrophoretic mobility of ovoinhibitor and a 2-proteinase inhibitor The difference in electrophoretic mobility between a2-proteinase inhibitor and ovoinhibitor was reminiscent of that between chicken serum transferrin and ovotransferrin, which is attributable to the presence of sialic acid residues in the serum protein, but not in that from egg white [4]. Ovoinhibitor (200 #g/ml), and a2-proteinase inhibitor (300/~g/ml) purified by affinity chromatography, were incubated with neuraminidase (100 units/ml) in 50 mM sodium acetate buffer, pH 5.5, containing 5 mM CaC12. Incubation was for 1 or 2 h at 37 °C. After incubation, the solutions were stored at --20 °C until 5-#1 samples were run in immunoelectrophoresis together with samples of the untreated antigens in the same buffer. The result of the experiment (Fig. 5) showed a marked reduction in the mobility of a2-proteinase inhibitor following the action of neuraminidase, whereas ovoinhibitor was practically unaffected, so that finally the electrophoretic mobility of a2-proteinase inhibitor was very similar to that of ovoinhibitor. The completeness of neuraminidase action under the conditions used was shown by the finding that the mobility of each protein was the same after either 1 or 2 h incubation. Proteinase inhibition by az-proteinase inhibitor The capacity of a2-proteinase inhibitor to inhibit trypsin, chymotrypsin and Aspergillus proteinase was tested by use of the protein purified by affinity chromatography. The assay methods were as described in Materials and Methods. Trypsin was assayed with a-N-benzoyl-o,L-arginine p-nitroanilide as substrate. It was found that 42 ~o inhibition of 4.9 #g of trypsin was produced by 3.75/tg of a2-proteinase inhibitor, and 93 ~ by 11.25 #g. Chymotrypsin was assayed with azo-casein as substrate. It was found that 39 ~ inhibition of 8 #g of enzyme was produced by 7.5 #g of a2-proteinase inhibitor, and 69 ~o inhibition by 38 #g. Aspergillus proteinase was assayed with azo-casein as substrate, and inhibition of 50/~g was 61 ~ by 1.5/~g, and 73 ~ by 15/~g. It seems likely that the resistant

60

Fig. 5. The effect of neuraminidase on the mobility in immunoelectrophoresis of a2-proteinase inhibitor and ovoinhibitor. The wells contained (i) a2-proteinase inhibitor (1.5/zg), (ii) a2-proteinase inhibitor treated with neuraminidase (see Materials and Methods) for 2 h, (iii) ovoinhibitor (1.0/tg), (iv) ovoinhibitor treated with neuraminidase for 2 h, and (v) chicken serum (1/d). All of the troughs contained anti-(ovoinhibitor) serum. activity was due to the EDTA-sensitive proteolytic enzymes o f A. oryzae [15, 16], whereas the serine proteinase [17] was readily inhibited. It was concluded that a2-proteinase inhibitor shares the rather broad specificity o f ovoinhibitor for the inhibition o f serine proteinases. The results indicate that the affinity of the az-proteinase inhibitor for the enzymes was lower than that reported by others for ovoinhibitor, however. The activity of a2-proteinase inhibitor after elution from the affinity column at p H 3 shows that it is a more stable proteinase inhibitor than those that have been recognised in mammalian sera. Of course, the characteristics o f the purified a2-proteinase inhibitor could be those of a "modified" rather than a "virgin" inhibitor, and it is not yet clear how important this distinction may be for ovoinhibitor [18].

Attempted detection of ct2-proteinase inhibitor in mammalian sera Rabbit anti-(ovoinhibitor) serum was run in double immunodiffusion against sera o f rabbit, sheep, pig and man. N o reaction was detected. It was concluded that if these mammalian sera contain a protein h o m o l o g o u s with ovoinhibitor it is too dissimilar to react with the antiserum. M a n y serum proteins of chicken undoubtedly are h o m o l o g o u s with those of mammalian sera, and yet do not cross-react, so this result was not considered very surprising.

61 DISCUSSION Having a molecular weight of about 50 000 and the property of inhibiting both trypsin and chymotrypsin, ovoinhibitor bears a superficial resemblance to human al-trypsin inhibitor. It was for this reason that the possibility of the presence of ovoinhibitor in chicken serum was examined, initially. The serum form of ovoinhibitor, az-proteinase inhibitor, that was discovered seems to have the necessary properties for a physiologically significant proteinase inhibitor, but differs from known mammalian inhibitors. The properties and composition of chicken ovoinhibitor have been carefully studied [2, 19], but the results of gel gradient electrophoresis in the present work were unexpected. Ovoinhibitor ran in the gel gradients as if it had a molecular weight of about 70 000, rather than 50 000. Presumably, the presence of carbohydrate (6-10 ~ ; see ref. 2) may have had some effect; an explanation which seems particularly plausible since multiple forms of ovoinhibitor were resolved, and these have been shown to differ primarily in their carbohydrate content [2]. The possibility that the heterogeneity of ovoinhibitor arises as an artefact of the exposure to low pH in the procedure of Matsushima [1] is excluded by the work of Davis et al. [2], who used much milder methods. The serum form of ovoinhibitor showed greater anodal electrophoretic mobility than ovoinhibitor itself. This suggested a striking analogy with the ovotransferrintransferrin system as elucidated by Williams [4, 5]. The latter proteins were found to have identical polypeptide structures, but to differ in the composition of the oligosaccharide prosthetic group. The carbohydrate moiety of transferrin contained one or two residues of sialic acid, whereas that of ovotransferrin was composed entirely of neutral sugars, and it was shown that this difference could account for the slightly greater electrophoretic mobility of transferrin. The egg white protein was not derived from serum transferrin by a secondary modification, for the oviduct was shown capable of the de novo synthesis of ovotransferrin. A single structural gene controlled the synthesis of both proteins, however. In the present study, it has been found that the mobility of a2-proteinase inhibitor is reduced approximately to that of ovoinhibitor by exposure to neuraminidase. Ovoinhibitor itself seemed unaffected as was to be expected, since it contains only 0.2-0.5 residues of sialic acid per mole [2]. It may well be that the differences between ovoinhibitor and a2-proteinase inhibition are confined to the carbohydrate moiety, as was found for the transferrins. The immunological identity shown within each pair of proteins indicates that the carbohydrate structures do not contribute significantly to antigenicity. Chicken ovoinhibitor has proved to be an efficient immunogen, and it was surprising that the two commercial antisera to chicken serum proteins did not contain antibodies reactive with serum ovoinhibitor or ovoinhibitor itself. One possible explanation would be that serum ovoinhibitor is not precipitated by the potassium aluminium sulphate procedure commonly employed in the preparation of "whole serum proteins" as immunogens. The finding that az-proteinase inhibitor exists in the serum of cockerels at a concentration similar to that in hens shows that its synthesis is not linked to the production of egg white. On the contrary, it raises the possibility that the protein may first have been evolved as a plasma protein, as is presumably also true for transferrin,

62 and later adapted to use in the egg. The concentration of a2-proteinase inhibitor in chicken blood (1 mg/ml) was found to be the same as that o f ovoinhibitor in egg white, and is quite high enough to be consistent with a physiological role of the protein in the control o f proteolytic activity. The binding of trypsin and c h y m o trypsin by az-proteinase inhibitor is strongly reminiscent o f the activity of al-trypsin inhibitor in h u m a n serum, and there seems a g o o d possibility that the two inhibitors are homologous. Recent work [20] indicates that al-trypsin inhibitor has duplicate binding sites for trypsin, contrary to the previously accepted view. The duplicate binding sites ovoinhibitor possesses for trypsin and chymotrypsin [2] are presumably shared by a2-proteinase inhibitor, a2-proteinase inhibitor differs from a r t r y p s i n inhibitor in its lower electrophoretic mobility, and m u c h greater stability to heat and acidic conditions, but these differences might be accounted for by relatively small variations in structure. Ovoinhibitor is an exceptionally interesting natural inhibitor of proteinases, because of its broad specificity and multiplicity of binding sites. The discovery of its h o m o l o g u e in chicken serum adds to our knowledge of the proteinase inhibitors of blood. It will be of interest to establish definitely the relationship of chicken a2-proteinase inhibitor to a : t r y p s i n inhibitor, or possibly some other inhibitor in m a m malian sera. ACKNOWLEDGEMENTS I thank Mrs Molly Brown for her excellent technical assistance, and my colleagues for much helpful comment. REFERENCES 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20

Matsushima, K. (1958) Science 127, 1178-1179 Davis, J. G., Zahnley, J. C. and Donovan, J. W. (1969) Biochemistry 8, 2044-2053 Gertler, A. and Feinstein, G. (1971) Eur. J. Biochem. 20, 547-552 Williams, J. (1962) Biochem. J. 83, 355-364 Williams, J. (1968) Biochem. J. 108, 57-67 Porath, J., Aspberg, K., Drevin, H. and Ax6n, R. (1973) J. Chromatogr. 86, 53-56 Erlanger, B. F., Kokowsky, N. and Cohen, W. (1961) Arch. Biochem. Biophys. 95, 271-278 Chase, T. and Shaw, E. (1967) Biochem. Biophys. Res. Commun. 29, 508-514 Charney, J. and Tomarelli, R. M. (1947) J. Biol. Chem. 171, 501-505 Mancini, G., Carbonara, A. O. and Heremans, J. F. (1965) Immunochemistry 2, 235-254 Hjert6n, S. (1962) Arch. Biochem. Biophys. Suppl. 1, 147-151 Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134 Neville, D. M. (1971) J. Biol. Chem. 246, 6328-6334 Feinstein, G. (1971) Biochim. Biophys. Acta 236, 74-77 Nakadai, T., Nasuno, S. and Iguchi, N. (1973) Agric. Biol. Chem. 37, 2695-2701 Nakadai, T., Nasuno, S. and Iguchi, N. (1973) Agric. Biol. Chem. 37, 2703-2708 Nakadai, T., Nasuno, S. and Iguchi, N. (1973) Agric. Biol. Chem. 37, 2685-2694 Feinstein, G. and Gertler, A. (1972) Eur. J. Biochem. 31, 25-31 Zahnley, J. C. and Davis, J. G. (1973) Biochem. J. 135, 59-61 Johnson, D. A., Pannell, R. N. and Travis, J. (1974) Biochem. Biophys. Res. Commun. 57, 584-589