Conformation and sequence dependent antigenic determinants in human low molecular weight kininogen

Molrcuiar

I~?~rwriologr. Vol. 20. No. 6, pp. 669-678. 1983

0161-5890,8353.00+.00

Printed in Great Britain.

0 1983 Pergamon Press Ltd.

CONFORMATION AND SEQUENCE DEPENDENT ANTIGENIC DETERMINANTS IN HUMAN LOW MOLECULAR WEIGHT KININOGEN ANN-CHRISTINE Department

SYVANEN, of Biochemistry.

TYTTI University

K_&RKK#INEN of Helsinki,

and

ULLA

SF-001 70 Helsinki

HAMBERG 17, Finland

Abstract-Conformation and sequence-dependent antigenic determinants were investigated using a kinin-free low molecular weight kininogen isolated from Cohn’s plasma fraction IV. This antigen contains the determinants of the apparently intact heavy chain common to the high molecular weight and low molecular weight kininogens. Straightforuard reduction and carboxymethylation destroyed the immunoreactivity of this molecule. Antiserum prepared against the reduced protein recognized both reduced and unreduced antigen showing the presence of both types of antigenic determinant. The corresponding antibodies uere separated using immunoadsorbent columns. As shown by the higher avidity of the antibodies. the conformntlon-dependent determinants dominate the antigenic structure.

INTRODUCTION

is a single plasma glycoprotein kininogen chain polypeptide molecule containing the covalently bound vasoactive kinin segment and functions as a substrate for kallikreins (E.C. 3.4.21.8) (for ref. see Pisano & Austen, 1974). Human plasma contains two kininogen molecules, the HMT (120,000 mol. wt) and LM, (60,000 mol. wt) kininogen, that differ by the structure and function of the light chain, which can be separated from the heavy chain after removal of kinin and reduction (Kerbiriou & Griffin, 1979). It has been found earlier that these molecules are immunologically related (Habal rt al., 1975; Kerbiriou et N/., 1980). In an earlier study we presented evidence that the antigenic determinants shared by HM, and LM, kininogens are located in the heavy chain (Syv;inen et al., 1981). So far any further relationship between these molecules is unknown. It would therefore be of importance to know the structure of the heavy chain which apparently is a common denominator of both molecules. In general proteins contain both conformation and sequence dependent determinants. It is generally assumed that antibodies to native The

*Abbreviations: HM,. high molecular weight; LM,, low molecular weight: H, antigen. kinin-free LM, kininogen isolated from Cohn’s plasma fraction IV; RCM, reduced and carboxymethylated: RIA. radioimmunoassay; SDSPAGE. sodium dodecyl sulphateepolyacrylamide gel electrophoresis; HPLC. high performance liquid chromatography.

proteins are directed mainly against conformational rather than sequential determinants (Sela et al., 1967, Kabat. 1980). In the present work the antigenic determinants of human LM, kininogen were further studied. For this purpose we have utilized a kinin-free LM, kininogen purified from Cohn’s plasma fraction IV (Kirkkginen et Al., 1982). Antiserum against this antigen has been shown to recognize the native kininogens in normal human serum. We have investigated the effect of conformational change on the immunoreactivity of kininogen and found that the antibody combining sites consist of both conformational and sequential determinants in the heavy chain.

MATERIALS

AND

METHODS

Materids

Normal blood bank plasma (anticoagulant citrate-phosphate-dextrose) and Cohn’s plasma fraction IV paste was obtained from the Finnish Red Cross Blood Transfusion Service (by the courtesy of Dr. G. Myllylg). Single donor plasma samples (anticoagulant 0.8% citric acid, 2.2”/, trisodium citrate, 2.45x1 dextrose, 1 mli9 ml blood) were obtained from healthy volunteers. Commercial antisera against human plasma proteins were from Behringswerke AG, or Dako Immunoglobulins Ltd. Copenhagen and antinormal human serum (OOHP) from Organon Teknika. Holland. Sheep anti-rabbit IgG was a

670

ANN-CHRISTINE

SYVANEN.

TYTTI

gift from Professor C.-G. Gahmberg, University of Helsinki. Human sc,HS-glycoprotein (op 10671) was from Behringwerke AG, ceruloplasmin and albumin were from Kabi AB. Stockholm. r.zHS-glycoprotein and albumin were immunologically pure by immunodiffusion analyses. Trypsin. EC 3.4.21.4 (TRTPCK, 31 N 840) and soy bean trypsin inhibitor (crystalline) were from Worthington, Freehold. NJ, and staphylococcal serine proteinase (V 8 protease) EC 3.4.2 1.19 from Miles Biochemicals, Elkhart. IN. Na’““I (IMS 30, carrier-free) and iodo-(2‘“C)-acetic acid were from the Radiochemical Centre, Amersham. U.K. Acrylamide and .v’. !V’-methylenebisacrylamide (specially purified for electrophoresis) from BDH Chemicals Ltd. Poole and Serva Blue@ from Serva Feinbiochemica, Heidelberg. Tyr’-bradykinin was from New England Nuclear. Boston. MA and bradykinin triacetate from Sigma Chemical Co.. St. Louis. MO. Urea and guanidineHC1 (ultrapure) were purchased from Schwarz,!Mann Inc.. Spring Valley. NY, dithiothreitol (A grade) from Calbiochem, San Diego, CA, cyanogen bromide from Eastman Kodak Co.. Rochester. NY, Freund’s complete adjuvant from Difco Laboratories. Detroit, MI, and acetonitrile (Lichrosorb) from Merck AG. Darmstadt. All other reagents were of analytical purity.

HM, and LM, kininogens were isolated from fresh human plasma (400 ml) by chromatography on QAE-Sephadex A-50 as described before (Hamberg et al., 19826). HM, kininogen was collected after elution at 0.35 M NaCl. The LM, kininogen was obtained from the effluent fractions before starting the salt elution. The effluent pool contained on average 96”; of the total plasma LM, kininogen. The effluent pool was submitted to the purification procedures described in detail in an earlier publication (Hamberg et al., 1975) using 1.9 M ammonium sulphate precipitation, DEAE-chromatography and Sephadex G-200 gel filtration. Immunologically pure kininogen was obtained by removal of impurities on a mixed immunoadsorbent column containing antibodies against ceruloplasmin and z,HS-glycoprotein (Karkkainen et (I/., 1982). An immunoreactive kinin-free LM, kininogen (referred to as the Hc antigen) was isolated from Cohn’s plasma fraction IV as described earlier (Karkkainen et ul.,

KARKKAINEN

and ULLA HAMBERG

1982). The same immunoadsorption was used to obtain immunologically kininogen.

method pure

Kininogen (1 -8 mg Hc antigen) was dissolved in 2 ml 0.2 M TrisHCI, 2 rnbf EDTA. 4M urea, pH 8.6. Dithiothreitol was added to 0.2 mA4 under nitrogen and reduction was carried out for 1 hr at 25°C. For carboxymethylation 2.5 Atmole iodoacetic acid containing 33 nmole iodo-(2-‘“C)-acetic acid (54 mCi. mmole), was added under nitrogen. After incubation for 1 hr at 25°C in the dark. excess of reagents were removed by dialyzing against two changes of 100 ml 0.1 M Tris-HCI, 1 miM EDTA, 4 M urea. pH 8.6. 100 ml of the same buffer without urea for 30 min in the dark and then against two changes of 400 ml H,O and finally against 400 ml 0.15 M NH,HCO,. pH 7.8 overnight or by gel filtration on a Sephadex G-10 column (0.8 x 5 cm) in 0.15 !LI NH,HC03. pH 7.8. The heavy and light chains were separated by gel filtration on an Ultrogel AcA 54 column (0.8 x 35 cm) in (I. 15 M NH,HC03, pH 7.8.

Radiolabelling of kininogen antigens and Tyr’-bradykinin was performed according to Greenwood rt ul. (1963) using chloramine-T. Free Na”‘I was removed by gel filtration on Sephadex G-25 and G-IO respectively, using 50 mM phosphate buffer pH 7.5 and a column The sp. act. washed with I”, bovine albumin. was on average 50 ,nCi//q of “‘I-kininogens and about 850 pCi/Llg for ““I-Tyr*-bradykinin. Radioimmunoassay (RIA) was performed as described earlier (Syvanen ct 01.. 1981). All dilutions were made in 50 mM Na-phosphate. 0.15 n/! NaCI. I”,, bovine albumin, pH 7.5. For estimation of kininogen antibody content serial dilutions of immunoglobulin preparations were incubated with labelled antigen substituting sample or standard with buffer. Average ahinity constants were determined by Scatchard analysis (Scatchard. 1949) of the RIA standard curves (Berson & Yalow, 1969). Radioactivity was measured in an LKB Wallac 1282 Compugamma counter. RIA of bradykinin was performed according to Talamo clr al. (1969) and Goodfriend & Odya ( 1974). Anti-bradykinin serum was prepared by immunization as described with

Antigenic

Determinants

1.0-I .5 mg doses of bradykinin coupled to ovalbumin using the toluene diisocyanate method (Talamo rt al., 1968). Kininogen (30-300 c(g immunoreactive kininogen) samples were incubated with 5 pg trypsin dissolved in 0.2 M Tris-HCI, 1 mM l,lO-o-phenanthroline, pH 8.0 for 30 min at 37°C. The reaction was terminated by addition of 5 pg soy bean trypsin inhibitor. Radioactivity was measured by liquid scintillation (Wallac 8 1000).

Monospecific antisera were raised in rabbits (male. 2-3 kg) using 0.1-0.2 mg of the antigen (LM,, HM,. H, kininogen, RCM-heavy chain) dissolved in 0.5 ml sterile 0.15 M NaCl emulsified with 0.5 ml Freund’s complete adjuvant injected (i.d.) biweekly (Hamberg & Tallberg, 1972. Kgrkkginen er al.. 1982). The titres were determined by single radial immunodiffusion according to Becker (1969).

Separation of antibodies against conformation und sequence-dependent determinants IgG (2.1 mg) isolated from the anti-H, antigen serum was chromatographed first on an affinity column (0.4 x 5.3 cm) prepared of RCM-heavy chain coupled to Sepharose 4B in 0.1 M Tris-HCI, 0.5 M NaCl, pH 8.0. The emuent was further passed through an affinity column prepared of native Hc antigen. Bound antibodies were eluted from each column with 6 M guanidine-HCl. Four 0.5 ml fractions were collected into test tubes containing 0.25 ml 0.15 M phosphate buffered saline, pH 7.5 and immediately dialyzed (2 x 2 hr) against 500 ml of the same buffer, and finally 48 hr against several changes of distilled water at 4°C. The same procedure was repeated with IgG (2.3 mg) isolated from the anti-RCM-heavy chain serum. The anti-kininogen activity at all stages of the experiment was assayed by RIA. Enzymatic

The immunoglobulin fractions of antisera were isolated by two successive (NH&SO4 at precipitations of 500;;) and 407; saturation pH 6.8. The precipitates were dissolved in Hz0 and dialyzed against 0.1 M NaHCO,, 0.5 M NaCI, pH 8.0, for preparation of immunoadsorbents. IgG from anti-H, antigen serum and antiserum against the heavy chain of reduced and carboxymethylated H, antigen (both obtained after 20 weeks of immunization) were further purified after dialyzing against 10 mM pH 8.0 by DEAE-cellulose Na-phosphate, (DE-52) chromatography according to Reif (1969). Preparation antigens

of’ .speci$c

invnunoadsorbents

with

Sepharose 4B was activated by CNBr according to Axtn et al. (1967). Kininogens (0.9 mg H, antigen and 2.8 mg heavy chain of reduced and carboxymethylated H, antigen respectively) were immediately coupled to 1.5 g activated Sepharose by gentle mixing for 2 hr at 25°C in 0.1 M NaHCO,, 0.5 M NaCI, pH was coupled). The 8.0 (937; of the protein immunoadsorbent was washed with coupling buffer, 1 M ethanolamine, pH 8.0 (30 min), and four times alternating with 0.1 M Na-acetate, 1 M NaCl, pH 4.0 and 0.1 M Na-borate, 1M NaCI, pH 8.7 and finally with 0.1 M Tris-HCl, 0.5 NaCI, pH 8.0 for use in immunoaffinity chromatography.

671

of Kininogen

cleavage

of Hc kininogen

Cleavage of 1.0 mg H, antigen was performed in 0.5 ml 0.1 M Tris-HC1, pH 8.0 with 10 ,ug trypsin and in 0.5 ml 0.05 M ammonium acetate, pH 4.0 with 30 ,ug S. uurezls V 8 protease at 37°C overnight. Analytical

methods

Double immunodiffusion analysis was used to test protein purity and monospecificity of antisera (Ouchterlony, 1948). Kininogen content was determined by single radial immunodiffusion (Mancini et al., 1965) using monospecific antisera prepared against LM, kininogens (Hamberg & Tallberg, 1972; Hamberg et al., 1975; Ksrkk;iinen et al., 1982). Normal blood bank plasma containing 0.26 mg/ml biologically active kininogen determined as the bradykinin-equivalent by bioassay on the isolated guinea-pig ileum was used as standard (Hamberg et al., 1978). SDS-PAGE was performed on Sy/, slab gels according to Laemmli (1970) or on 127; gels with a N’N’-methylenebisacrylamide/acrylamide ratio of 1 : I5 using cytochrome c and its CNBr-fragments as molecular weight markers. Gels were stained for protein with Serva Blue@. HPLC was performed on a Milton Roy apparatus equipped with a Waters Associates UGK injector on a Hibar RP- 18 (250 x 4 mm) column using a linear gradient of O-70”/;; CH,CN in 10mM sodium phosphate, pH 4.3 for separation of enzymatic cleavage products

612

ANN-CHRISTINE

SYVANEN.

TYTTI

K.&RKK;iINEN

and

ULLA

HAMBERG

double dilfusion anal) 51s ahowing purity and immunological identity ol’ LM, h~n~no~cn an till: human wxrn (,4-N IS): H, an tipWI. (a) 5 pg ol’ the protans assayed agamst 30 ~1 polyvalent anti-normal serum (diluted to a titre of 0.3 mg.ml) xsa)ed against 10 /II (b) 30 Pl anti-LM, and anti-Hc kininogen normal human serum (NHS). Fig

of H, kininogen. The light chain of H, kininogen was chromatographed on a Vydac TP 201 RP 10~ (250 x 4.6mm) column with a O-70?: gradient of 0.1 O;, trifluoroacetic acid in Hz0 to 0.1’3; trifluoroacetic acid in CH3CN. in preparation). (Lottspeich, F.. manuscript

Gradients were completed in 100 min at a flu\\ rate of 1.5 ml;min. Peptides were detected b! absorbance at 206 nm (Uvicord S 711X. LKB). These analyses were performed at the Max Planck Institute of Biochemistry tvith the kind help of Dr. Friedrich Lottspeich. Protein was determined by measuring the absorbance at 280 nm using Ai& nm = 10.0 and for pure kininogen preparations a value of 7.0. RESULTS

78000 68000 60000 43000

Fig. 2. XDS~polyacrylamide gel electrophorcsis (8”,, gel) comparing IO klg LM, (1). H, kininogen (2) and H, kininogen heavy chain (3) showing similar sire (60.000 mol. wt) and purity Molecular weight markers on the left.

As shown in Fig. 1. the prepared LM, and H, kininogens were immunologically pure b> double diffusion analysis using 5 Alg of the respective proteins. The apparent mol. wt was the same (60.000) for the two kininogens estimated by SDS-PAGE (Fig. 2). The amino acid contents are shown in Table I. The H,- antigen contained no or negligible bradykinin segment (co.05 pg/mg). The bradykinin content of LM, kininogen was I 1.6 pg;mg protein. Immunochemical identity of HM,. LM, and H,. kininogen is demonstrated in Fig. 3a showing the parallel RIA inhibition curves using anti-H, kininogen serum. The RIA inhibition curve ol and kininogens in normal the H, antigen human plasma were also identical (Fig. 3b). For experimental details see figure legends. The antigenic relationship between these kininogen antigens was further tested using the respective

Antigenic

Determinants

Table I, Amino acid composition (mole 4,)) of various human kininogens and the heavy and light chains of kininogen isolated from Cohn’s plasma fraction IV compared with the HM, kininogen heavy chain (taken from Nakayasu & Nagasawa, 1979)

Residue Asp Thr Ser Glu Pro Gly Ala Cys( I :2) Val Met Ile Leu TYr Phe Lys His Arg

H,

Hc H chain

13.6

13.3

8.2 1.4 IS.3 5.1 6.3 7.3 n.d. 4.5 I.3 3.7 7.5 3.9 5.1 7.6 2.1 3.9

8.5 9.6 13.4 2.8 7.9 7.4 2.8 4.1 I.3 3.6 6.6 2.8 3.9 6.2 2.6 2.5

Hc L chain

LM,

HM, H chain

4.6 6.1 13.5 18.5 2.6 9.1 8.5 2.7 3.1 0.7 3.6 4.2 3.0 I.4 6.7 2.4 7.3

10.2 x.2 8.6 14.9 3.6 6. I X.1 3.3 5.3 0.9 3.9 8.0 3.0 4.2 6.4 1.6 3.8

11.6 7.8 7.6 15.1 6.4 5.8 5.8 2.6 s.3 0.9 5.3 5.5 3.2 4.7 7.6 2.0 2.9

B/B0 1.0

of Kininogen

673

monospecific antisera. As demonstrated in Fig. 4 the HM,, LM, and H, kininogens are closely related antigens. The LM, and H, kininogens differed from HM, kininogen when assayed against the anti-HM, kininogen serum. This was clearly indicated by a slight spur formation due to the presence of antibodies against the HM, kininogen light chain.

The heavy and light chains of 8.1 mg Hc antigen were separated after reduction and carboxymethylation (Fig. 5). The approximate mol. wt of the heavy chain was 60,000 (Fig. 2) and 4500 for the light chain (not shown) by SDS-PAGE. The respective amino acid compositions are shown in Table 1. Two fractions of the light chain were separated by HPLC (Vydac TP 201 RP 10 p) (Fig. 6). The tripeptide N-terminal sequence of the first fraction was B/B,

-"\,0.-

1.0 b

.-

‘:.:-

1

10 ng

EKG

100

1 1:32

10 1:8

plasma

100

1:2 -32' dllutlon .lO

Fig. 3. Parallel radioimmunoassay inhibition curves produced by (a) HM, (O--U). LM, (P-O) and H, kininogen (t-0) (BKG); (b) Hc kininogen (+a) and kininogens in normal human plasma (*--*) measured against anti-H, antigen serum showing antigenic identitY of the molecules. B,‘B, is the ratio of 1251-labelled Hc antigen bound in the presence of inhibitor and absence of inhibitor.

Fig. 4. Double diffusion analysis of three kininogens (5pg) showing close relationship between the antigens assayed against (a) anti-H, kininogen serum (b) anti-LM, kininogen serum and (c) anti-HM, kininogen serum (30111 of each diluted to a titre of 0.3 mg/ml). Spur formation by HM, kininogen assayed against anti-HM, kininogen serum indicated by arrow.

674

ANN-CHRISTINE

elution volume ml

SYV,&NEN,

elution volume

TYTTI

ml

Fig. 5. Gel filtration on an Ultrogei AcA 54 column (0.X x 35 cm) of (a) 8.1 mg Hc antigen after reduction 3nd carboxymethylation. Excess reagents were removed prior to gel filtration by dialyzing; 5.3 mg heavy and 023mg light chain in the pooled fractions were collected (Ai& nm = 7.0); (b) 0.6 mg H, antigen carhoxymethylated in 4 M urea without prior reduction. ( -) Protein by LI.V.-absorhance. (O--O) immunoreactivc kininogen by single radial imm~jnodiffnsi~)n. (--. .) radioactivity in 5O~ll of each fraction.

K;iRKKalNEN

and

tILLi\

HAMBERG

Ser-Ser-Arg and the second Ile-Gly-Glu. The material was heterogeneous shown by the presence of other end groups. Due to lack oi material only an 11 amino acid sequence could be determined. As seen in Fig. 5a the immunoreactivit! measured by single radial immunoditrusion was abolished by reduction and carboxymeth>lation of the disulphide bonds. Amino acid analysis of the isolated heavy chain (RCMheavy chain) indicates that the carbouymethylation of cystein residues was complete. When H, antigen was submitted to carboxymethylation without reduction no radioactivity was incorpor~ited into the molccuie as iIlLlstr~lted in Fig. 5h. This suggests absence of fret sulphydry1 groups in the molecule. The number 01 disulphide bonds of the I-I, antigen was cstimated to five from the amount of “C’-labclled iodoacetic acid bound to the molecule (Fig. %I). The reduced and carbosymethyl~~tcd heav> A206

%B -70 d

nm

------:

I’

,’

-60

1

,’

1 i

_,’ I

-50

_,’

I

I’ ,’

-40

\

Fig. 6. Separation by high performance liquid chromatography of two peptides of the iight chain obtained after reduction and carhoxvmethviation of the H, antigen with different !v-termlnaf tripcptidc . , sequences. The undecapeptidc sequence of the first peptide‘is given. (- ----) gradient. ( experimental details see methods).

0

I

5

IO3 ng BKG

104

1 ~

“q

BKG

Fig. 7. Radioimmunoassay inhibition curves produced by H, antigen (0 ~~~ 0) and its heavy chain obtained after complete reduction and carboxymethylation (o- --0) using antiserum against (:i) H, I-Iv antlgcn bound in the presence of antigen and (b) Its heavy chain. B/B, is the ratio of “51-labelled to I I np H, antigen is inhibitor and absence of inhibitor. 5000ng (A 2R0 ..,) heavy chain compared needed for SW’;, inhibition of binding using anti-HC kininogen serum. The corresponding amounts \\ith the ~~ntisertiltl against the heavy chain were 75 ng :md 44 ng rccpccticcly.

Antigenic

Determinants

% bound ‘OOr

.._

10-l

a

10-2 10-3 10-4 antiserum dilution

10-S

675

of Kininogen % bound 100 r

ro-6

10-2

b

10-3 10-4 lo-5 antser”m dllutmn

Fig. 8. Comparison of the radioimmunoassay titration curves of antiserum against HM, (O--O) and LM, kininogen (*_o), H, antigen (-0) and the H, antigen heavy chain obtained after reduction and carboxymethylation (0~~0) using (a) ‘Z51-labelled Hc antigen and (b) ‘2”I-1abelled heavy chain.

chain lost most of its response in RIA against the H, kininogen antiserum as illustrated in Fig. 7a in comparison with the unreduced protein. The amount causing 50% inhibition of binding of “51-labelled antigen was about 400 times larger than the amount of the unreduced antigen. On the other hand reduction and carboxymethylation did not influence the antigenie response by RIA when the antiserum prepared against the RCM-heavy chain with destroyed conformation was used (Fig. 7b). Radioimmunoassay antibody titration curves of antisera against the various native kininogens were similar but differed in shape compared with the titration curve of the antiRCM-heavy chain serum assayed using the radio-labelled native antigen (Fig. 8a). The titration curves of antisera against H, antigen and the RCM-heavy chain are similar when labelled RCM-heavy chain is used (Fig. 8b). The average association constants of these antisera are shown in Table 2. The tryptic and S. aureus V 8 protease cleavage products of H, kininogen were submitted to HPLC (Hibar RP-18). About 50 peptide fractions of each digest were collected and assayed by RIA using anti-H, kininogen serum and anti-RCM-heavy chain serum. The cleaved material was positive in double immunodiffuTable 2. Association constants of anti-H, kininogen serum (A-H, kininogen) compared with the antiserum prepared against the heavy chain of H, antigen obtained after reduction and carboxymethylation (A-RCM-heavy chain) using reduced and unreduced antigen Antiserum A-H, kininogen A-H, kininogen A-RCM-heavy chain A-RCM-heavy chain

Antigen H, kininogen RCM-heavy chain H, kininogen RCM-heavy chain

K, W

‘)

3.8 IO” I.1 I06 4.5. 108 3.0.108

sion before fractionation by HPLC but no immunoreactivity was detected in the separated peptide fractions. Separation

of antibodies

and sequential

against

determinants

conformational

by ajinit~~ chrorna-

tography

As illustrated in Fig. 9 (1) X0;/, of the anti-H, kininogen antibodies passed unbound through the RCM-heavy chain column, while only negligible activity (
A-RCM-heavy

IgG

‘gG

cham

~ ,!+ ‘.,_I

,:.\’ ,+

I

&

RCM-

7 Q

0

0,

:

ad

heavy chain

I 0 j.

1

2

3

Fig. 9. Schematic representation of the procedures used to separate antibodies against conformational and sequential determinants using immunoadsorbent columns (0.4 x 5.3 cm) (for experimental details see methods). Purified IgG from anti-H, antigen serum was chromatographed on a column (1) prepared with RCM-heavy chain protein (0) SO’%;of the antibody activity (RIA) passed through (vertical arrows) and subsequently put through a column (2) prepared with H, antigen protein (0). All antibody activity was retained (
676

ANN-(‘HRISTINE

SYV;iNEN.

TYTTI

%bound 100

40

5.0

0.63

0.078

iJQ'@ (A280 nm)

Fig. 10. Radioimmunoassay titration curves of the antibodies eluted according to the procedures in Fig. 9. Antibodies against sequential (O-O) and conformational (0-O) determinants in the anti-H, antigen serum and antibodies against sequential determinants (o--e) in the antiserum against the H, antigen heavy chain obtained by reduction and carboxymethyiation.

the antibody activity was found in the effluent of the RCM-heavy chain column in Fig. 9 (3). In Fig. 10 the titration curves of the antibodies eluted (lo-20% of the antibodies bound) with guanidine-HCl are shown. The average association constants measured with the H, antigen of the eluted antibodies are shown in Table 3.

DISCUSSION

The H, antigen is well suited for studies of the antigenic determinants of the kininogen heavy chain due to its immunologic~~l identity with HM,, LM, and the native plasma kinino&ens. Only when assayed against antiserum to HM, kininogen the different antigenic structure of the HM, kininogen light chain appears, which can be clearly seen as a spur formation in double immunodiffusion analysis. Furthermore, using the H, antigen separation of the heavy and light chain is experimentally easy to perform due to the complete lack of kinin segment. The present findings demonstrate that the H, kininogen contains both conformational

Table 3. Association constants of antibodies against conformational and sequential determinants of the H, antigen separated by immunoadsorption determined using the H, antigen -.._ ..__ K, of antibodies

“The numbers

cluted

from immunoadsorbent

I. X.‘~IV !K2. _._ 7 7. 10” AC

’

3. 7.0. IOX M

/

represent

experiments

shobvn in Fig <>

KaRKK;iiNtN

and

l!LLA

HAMBERG

and sequenti~~l antigenic deterrnill~~llts. Atassi and coworkers have demonstrated that serum albumin contains four antigenic sites of the sequential and two sites of the conformational type (Atassi, 1987: Sakata & Atassi. 19X0). Similarly antibody against c;trcil~oerlibryonic antigen recognized both conformation and sequence dependent determin~~nts of the molecule ~Sundbl~~d PI u/., 1979). As shown bq. the respective association constants of the H,. kininogen antiserum against the native. unreduced antigen has the highest affinity for the conformation dependent combining sites. which \vas further ascertained by determination of the association constants of the separated antibodies against conformational and sequential determinants. The higher association constant shows that the avidity of the antibodies is greater for the unreduced than for the reduced and denatured protein lacking the structure depending on intact disulphide bonding. The importance of the single disulphide bond linking the heavy and light chains for the conformational combining sites is unknown. It seems possible by the high degree of heterogeneity found so far with the H, antigen light chain that its peptide structure prr sr may not be decisive for the conformation dependent antigenie structure of the heavy chain. This is further sustained by the immunological identity of all forms of kininogeI1 inclLldi1~~ the HMr with its nntigenically specitic light chain. Despite the ten times higher association constant against the conformational determinants it is obvious that the avidity of the antiserum prepared against the reduced and c:lrboxymethylated antigen is still high. This antiserum reacts identically with the native and the reduced antigen. which suggests the presence of a major antigenic site of se~lLlellti~iil~ arranged amino acids, which is independent of the nati\‘e conformation. So far our attempts to find ;t major antigenic site after proteinase degradation of the H, antigen have failed. This ma> depend either on incomplete digestion with trypsin and the V 8 proteinase or on the necessity to bring several larger peptide sequences together. Kerbiriou rt a/. (1980) have prepared antisera against reduced and carboxymcthylated light and heavy chains of HM, kininogcn. These antisera representing antibodies against sequential determinants of the molecule were specific for the light chain of HM, k~~lino~eti and the shared heavy chain of HM! and LM,

Antigenic

Determinants

kininogen respectively. As ascertained by present findings our antiserum, however, prepared against the unreduced H, antigen can be used for quantitative determination of all native forms of kininogen. Due to the higher association constant of the antiserum against the conformation dependent determinants this should be the method of choice for determination of kininogen in do. This was also the approach used by Proud et al. (1980) who determined HM, kininogen using an antiserum specific for HM, kininogen prepared by removal of antibodies common to all kininogens by immunoadsorption. An interesting result by Kleniewski (1979) suggests that plasmin uncovers a new antigenic site common to HM, and LM, kininogen and a site specific for HM, kininogen. In our study we did not find exposure of any new antigenic determinants by reduction and carboxymethylation. The Hc antigen used has been subject to degradation by activated proteinases in the Cohn fractionation procedure. Comparatively high plasma kallikrein activity was measured in the starting material using the chromogenic peptide H-D-Pro-Phe-Arg-pNA (Karkkainen et al., 1982). It is not likely that kallikrein activation in human plasma exposes hidden sequential determinants more dominating than conformation-dependent determinants the measured by the antiserum against the H, antigen as indicated in recent studies (Hamberg et LA.. 1982). In an earlier study (Hamberg et a/., 1978) we compared the immunochemically determined kininogen in synovial fluid using monospecific anti-LM, kininogen serum with the kininogen calculated from the determination by bioassay of the bradykinin equivalent to show that bradykinin had been released in human synovial fluid from inflammatory rheumatoid arthritis. Similarly the immunochemically determined kininogen increase in the inflammatory response together with other acute phase proteins did not correspond to the kininogen determined by bioassay, suggesting release of the vasoactive bradykinin fragment (Hamberg et al., 1982a). Present findings sustain that the monospecific antiserum against the easily available kinin-free kininogen antigen purified from plasma fraction IV can be applied for immunochemical assays of various molecular forms of human plasma kininogens provided that conformation-dependent determinants of the heavy chain are present.

of Kininogen

677

Acknow[edyements-This investigation was supported by grants from the Magnus Ehrnrooth Foundation (A.-C.S.) and from the Signe and Ane Gyllenberg Foundation and Societas Scientiarum Fennica (U.H.) Valuable assistance was given by Ms. Mirja Kurki. The authors would like to acknowledge the help by Dr. A. Henschen-Edman and Dr. F. Lottspeich at the Max Planck Institute of Biochemistry. Martinsried, Munich, in performing the amino acid and sequence determinations.

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