The Conformational State of Human α2-Macroglobulin Influences Its Dissociation into Half-Molecules by Sodium Thiocyanate

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Vol. 333, No. 1, September 1, pp. 35–41, 1996 Article No. 0361

The Conformational State of Human a2-Macroglobulin Influences Its Dissociation into Half-Molecules by Sodium Thiocyanate Vithaldas P. Shanbhag,* Torgny Stigbrand,† and Poul Erik H. Jensen†,1 *Department of Biochemistry and †Department of Immunology, Umea˚ University, S-901 85 Umea˚, Sweden

Received March 25, 1996

Sodium thiocyanate dissociates native human a2macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2 M. Incubation with 1.6 M sodium thiocyanate for 1 h at 227C dissociates about 90% of a2-macroglobulin into half-molecules. The halfmolecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2 M or zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the ‘‘closed-trap’’ than in the ‘‘open-trap’’ conformation of a2-macroglobulin. The cleavage in the bait region by trypsin makes a2-macroglobulin completely stable toward dissociation, and a2-macroglobulin remains in a tetrameric state in 2.2 M sodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation. q 1996 Academic Press, Inc. Key Words: human a2-macroglobulin; half-molecules; dimer–dimer interaction; sodium thiocyanate.

A large number of species contain in their plasma high-molecular-weight glycoproteins some of which are a-macroglobulins, which occur as monomers, dimers, or tetramers (1). In humans two a-macroglobulins, a2M2 and PZP, have been found. Human a2M is a tetra1 To whom correspondence should be addressed. Fax: /46-90102250. 2 Abbreviations used: a2M, human a2-macroglobulin; PZP, pregnancy zone protein; MeNH2 , methylamine; MMTS, methyl methanethiosulfonate; NaSCN, sodium thiocyanate; a2M–MeNH2 , a2M treated with methylamine; a2M–MeNH2 –SCH3 , a2M treated with methylamine and MMTS; a2M–MeNH2 –SCH3 –trypsin, a2M– MeNH2 –SCH3 treated with trypsin.

mer of four identical subunits, each of 180 kDa in molecular mass, and the native protein is assembled by the noncovalent association of two disulfide-bonded dimers (2, 3). It is a unique extracellular proteinase inhibitor with the capacity to sterically inhibit proteinases of all types by a process termed ‘‘trapping,’’ which is initiated by a cleavage, by the proteinase, in the proteolytically sensitive region termed the ‘‘bait’’ region of the a2M-subunit. A maximum of two proteinase molecules can be inhibited by a2M (4, 5). The trap closure in a2M involves the cleavage of internal b-cysteinyl-gglutamyl thiol esters, one in each subunit of a2M, and causes large conformational changes detectable by PAGE. Exposure of the receptor recognition sites follow the cleavage of the thiol esters and the ensuing conformational changes (6). Certain primary amines such as methylamine can cleave the thiol ester and bind to the exposed carboxylate residue. This also results in trap closure and exposure of the receptor recognition sites (7). We have previously shown that trapping in a2M is regulated at the thiol esters, in contrast to its dependence on bait-region cleavage in PZP (8). A striking difference between the native state of the two human a-macroglobulins is that the PZP molecule consists of a single disulfide-linked dimer with a molecular mass of 360 kDa. Even though PZP forms tetrameric species upon binding of proteinases, we have not been able to detect hydrophobic contact surfaces holding the PZP– tetramer together. The PZP–tetramer is probably formed by the covalent binding of the proteinase (8–11). The dimer–dimer contact in a2M might play an important role in the regulation of the conformational changes and trapping (9). Our knowledge about this contact surface and its function is, at present, limited. One approach to study the noncovalent interactions between the two dimers in a2M is to elucidate the factors which cause the dissociation of the tetramer into 35

0003-9861/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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half-molecules and to characterize the latter. As illustrated by Feldman et al. (12) there are two distinct ways in which the subunits can be linked to yield the functional a2M. Two types of half-molecules are conceptually possible. One type is obtained by reduction of the intersubunit disulfide bonds and the other by disruption along the noncovalent axis (13). Half-molecules obtained by reduction of intersubunit disulfide bonds (14, 15) retain proteinase binding activity and also yield the tetramer on reoxidation of the disulfide bonds (16). Dissociation of a2M along the noncovalent axis by urea or guanidine hydrochloride or SDS (13, 17–21) or low pH (22) has been claimed to generate denatured and nonactive half-molecules (21, 22). There are however reports that urea-induced half-molecules retain kallikrein (18) and trypsin (13) binding activity. It has also been reported that active half-molecules can be obtained by dissociation of a2M at very low concentration (õ1007 M) in 10 mM CdCl2 (23). These reports have led to the proposal that active half-molecules can be obtained either by reduction of the intersubunit disulfide bonds or by disrupting the noncovalent interaction between the half-molecules (13). In the present work we have studied the effect of sodium thiocyanate (24) on native a2M and its derivatives with trypsin, succinylated trypsin, and methylamine without or in combination with MMTS, as well as the derivative obtained by treatment of a2M– MeNH2 –SCH3 with trypsin. This permitted us to investigate the dependency of the dissociation of a2M on its conformation. Furthermore, the kinetics of the changes in fluorescence of a2M incubated with 1.6 M NaSCN has also been determined. MATERIALS AND METHODS Chemicals and enzymes. All buffer substances and salts used were of highest purity. Methylamine, phenylmethylsulfonyl fluoride and bovine trypsin (type XIII) were obtained from Sigma Chemical Co. (St. Louis, MO). Trypsin (EC 3.4.21.4) was 90% active as determined by active-site titration with p-nitrophenyl-p*-guanidinobenzoate (25). The concentration of trypsin was calculated using a value of 15.4 for (E1%)1cm and 23.3 kDa for the molecular mass (26). The solvent for trypsin was 1 mM HCl/10 mM CaCl2 . Succinylated trypsin was prepared according to Ref. (27). Reducing SDS–PAGE showed that the succinylated trypsin so prepared cleaved in the bait regions of a2M, but did not bind to it covalently. MMTS was from Aldrich (Steinheim, Germany). Purification of a2 M. The protein was purified from frozen human plasma according to the method of Imber and Pizzo (28). The purity of the preparation was checked by gel electrophoresis and it was kept frozen at 0707C not more than 2 months before use. The solvent for a2M, unless otherwise indicated, was 0.1 M Na–phosphate buffer, pH 8.0 (buffer P). The concentration of a2M was determined from the absorption at 280 nm using a value of 8.9 for (E1%)1cm and 720 kDa for the molecular mass (29). The preparation of the derivatives of a2 M. All derivatives of a2M were prepared in buffer P. a2M–MeNH2 was obtained by incubating a2M with 0.4 M methylamine for 1 h at 227C. The excess reagent was removed by gel filtration on a NAP-5 column (Pharmacia Biotech,

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Sweden). a2M–MeNH2 –SCH3 was prepared by treating a2M with 0.4 M methylamine and 100 mM MMTS for 1 h at room temperature after which the excess of reagents was removed by gel filtration on a NAP-5 column (30). Bait-region cleavage was achieved by incubation of a2M or a2M–MeNH2 –SCH3 with the required amount of trypsin or succinylated trypsin for 3 min after which the proteolysis was inhibited by treatment with an 100-fold molar excess (to the proteinase) of phenylmethylsulfonyl fluoride. The analysis of baitregion cleavage was performed by SDS–PAGE under denaturing conditions on 7% gels. The samples were incubated for 90 min in 1% (w/v) SDS and 5% (v/v) a-mercaptoethanol at room temperature. Effect of NaSCN on a2 M and its derivatives. This was determined by incubating a2M and its derivatives with a known concentration of NaSCN in buffer P for the required time at 227C. The sample was then diluted with buffer P so that the concentration of NaSCN became 0.2 M. The extent of dissociation was determined by electrophoresis under nondenaturing conditions on a 4–15% gradient gel (Bio-Rad) using a2M in 0.2 M NaSCN (added immediately prior to electrophoresis) and a2M incubated with 1% SDS for 90 min at room temperature, as markers for the position of the tetramer and dimer forms of a2M, respectively. The electrophoresis was run at 25–30 mA/gel for 10 h at 47C. Alternatively, the dissociation of a2M and its complexes was analyzed by PAGE under nondenaturing conditions on 5% gels. The degree of dissociation was calculated from the scan of the Coomassie brilliant blue-stained bands using a 2202 Ultrascan Laser Densitometer (LKB, Sweden). The results are given as stain of the dimer band as a percentage of the total stain in the dimer and tetramer bands together. The results are an average of three dissociation experiments at each salt concentration. Chromatographic determination of Stokes radius (Re). Size exclusion chromatography on a column (550 1 16 mm) of Sephacryl S-300 HR (Pharmacia, Sweden) was used to determine the Re of a2M and a2M half-molecules, respectively. Buffer P was used for equilibration of the column, as the solvent for all the proteins, and for their elution. The flow rate was maintained at 50 ml h01 and the absorbance of the eluant was monitored at 280 nm. Fractions, 1 ml in size, were collected and the absorbance at 280 nm, of the relevant fractions, was measured on a Beckman DU spectrophotometer. The entire chromatographic experiment was carried out at 47C. The distribution coefficient, KD , of each protein was calculated using the equation: KD Å (Ve 0 V0)/(VT 0 V0), where Ve is the elution volume of the protein and V0 and VT are the void and total volumes of the column, respectively. V0 was determined using a sample of Blue Dextran 2000 (Pharmacia, Sweden) and VT was calculated from the dimensions of the column. The column was calibrated using the gel filtration calibration kit for high-molecular-mass proteins (Pharmacia, Sweden) and bovine serum albumin (Sigma Chemicals, U.S.A.). Stoichiometry of thiol ester cleavage. This was measured as the thiol groups titratable with 5,5*-dithio-bis(2-nitrobenzoic acid) on reaction of a2M or a2M half-molecules, in 0.2 M NaSCN in buffer P, with methylamine or trypsin (31, 32). a2M or a2M half-molecules was incubated for 3 min with trypsin, at a molar ratio of 1 to 3, before the addition of 5,5*-dithio-bis(2-nitrobenzoic acid) and EDTA, so that the final concentrations of the latter two were 100 mM and 1 mM, respectively. The absorbance at 410 nm after 15 min was noted. The final concentration of methylamine was 0.4 M and it was added after the addition of 5,5*-dithio-bis(2-nitrobenzoic acid) and EDTA. In this case the change in absorbance at 410 nm was recorded for 60 min from the addition of methylamine. The stoichiometry, mol thiol groups released/mol protein, was calculated from the increase in absorbance, using an e Å 13,600 M01 cm01 for the thionitrobenzoic acid group released. The kinetics of change in intrinsic fluorescence on incubation of a2 M with NaSCN. The time course of the change in intrinsic fluorescence at 207C was followed by measuring the fluorescence in a Shimadzu RF-5000 spectrofluorimeter. The excitation wavelength (lex) was 275 and 289 nm, respectively, and the corresponding emis-

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DISSOCIATION OF a2-MACROGLOBULIN BY SODIUM THIOCYANATE sion between 320 and 360 nm was recorded automatically. Solutions of a2M and NaSCN in buffer P and at 207C were mixed in a semimicrofluorescence cuvette to obtain a final concentration of 0.05 mg/ml (when lex Å 275 nm) or 0.1 mg/ml (when lex Å 289 nm) of a2M and 1.6 M of NaSCN. The temperature was maintained at 207C by thermostating the cuvette housing during the measurement. The time of mixing was defined as t Å 0. The fluorescence spectra at known times were recorded and the difference (DF) between them and the spectrum at t Å 0 was analyzed automatically. The first order rate constant, k, for the change in relative fluorescence at 340 nm was calculated from the data using the GraFit program (Erithacus Software).

RESULTS

The results in Fig. 1A demonstrate that when native a2M is incubated for 30 min at room temperature with 1.0–2.0 M NaSCN, the protein starts dissociating into dimers as the concentration of the salt equals 1.2 M and that the proportion of a2M half-molecules increases when the concentration of the salt increases. However, the proportion of a2M half-molecules increases only slightly (87 to 94%) when the concentration of NaSCN increases from 1.6 to 2.0 M. We therefore used 1.6 M NaSCN to study the effect of the time of incubation on the dissociation of a2M. As shown in Fig. 1B the degree of dissociation increases from 87% after 30 min incubation to 89% after 60 min and there is no significant increase in the degree of dissociation when the incubation time is increased to 105 min. Incubation with 1.6

FIG. 1. Gradient gel electrophoretic analysis of the dissociation of a2M by NaSCN. (A) The effect of incubation with different concentrations of NaSCN for 30 min at 227C. The salt concentrations are in lanes: a, zero; b, 1.0 M; c, 1.2 M; d, 1.4 M; e, 1.6 M; f, 1.8 M; and g, 2.0 M. Lane h contains half-molecules obtained by incubation of a2M with 1% SDS for 90 min at 227C. n.d. denotes not determined. (B) The results of incubation with 1.6 M NaSCN for different time periods at 227C. The times (in minutes) are in lanes: a, zero; b, 30; c, 60; d, 75; e, 90; and f, 105. The values recorded at the bottom of each lane represent the degree (in percentage) of dissociation obtained by scanning of the gels as described under Materials and Methods. The electrophoresis was performed on 4–15% gradient gels.

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TABLE I

Thiol Groups Exposed on Treatment of a2M or a2M HalfMolecules (a2M-h) with Methylamine (MeNH2) or Trypsin [0SH] exposed Protein

Treatment

a2M a2M a2M-h a2M-h

MeNH2 Trypsin MeNH2 Trypsin

(mol/mol protein) a

(mol/mol subunit)

3.95 3.90 1.82 1.76

0.99 0.98 0.91 0.88

a The values recorded are each an average of three experiments differing by {0.05.

NaSCN for 60 min at 227C was therefore chosen for the preparation of a2M half-molecules. No change in the degree of dissociation could be observed when the concentration of the salt was reduced to 0.2 M by gel filtration. This indicates that a2M half-molecules are stable under these conditions. The results in Table I demonstrate that the thiol esters are intact after the dissociation of native a2M into half-molecules by NaSCN because mol thiol groups/mol subunit, released by treatment of the halfmolecules with either methylamine or trypsin, is nearly the same as that released from the native protein after the same treatment. The Stokes radius of a2M half-molecules obtained by dissociation of a2M with 1.6 M NaSCN is decreased from 9.4 to 7.0 nm for the native protein (Fig. 2). A comparison of the size of the half-molecules obtained by different means (Table II) shows that the present method described here yields half-molecules with a Re lower than but much closer to that of half-molecules obtained by dissociation of a2M with 4 M urea (Re Å 7.5 nm) than to that of half-molecules obtained by reduction of a2M with 0.5 mM dithiothreitol (Re Å 5.7 nm). Furthermore, the experimentally determined Re for PZP is the same as that for the a2M half-molecule. Only the derivative a2M–MeNH2 –SCH3 approached the behavior of native a2M when different complexes of a2M were incubated for 60 min with increasing concentration of NaSCN (Table III), while it is more difficult to dissociate a2M–methylamine compared to the native protein. Significant dissociation of a2M–methylamine occurs only at a concentration of NaSCN equal to or higher than 1.4 M and at each higher concentration of the salt, the proportion of the half-molecule form of a2M–methylamine is lower than that obtained by incubation of native a2M with the same concentration of salt. The most striking result is that complexes in which the bait regions have been cleaved by trypsin (as judged from SDS–PAGE of SDS-denatured and reduced samples), a2M–trypsin and a2M–MeNH2 –SCH3 –trypsin, M

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FIG. 2. Chromatographic determination of Stokes radius, Re , of a2M, a2M half-molecules, and PZP, respectively. a2M (a), a2M halfmolecules (b), and PZP (c) were applied to a column (550 1 16 mm) of Sephacryl S-300 HR. The void volume of the column was determined using blue Dextran 2000. The column was calibrated with following standard proteins with their respective Re values in nanometers denoted within parentheses: 1, thyroglobulin (8.6); 2, ferritin (5.9); 3, catalase (5.2); 4, aldolase (4.6); and 5, bovine serum albumin (3.5). The determination of KD of each protein was as described under Materials and Methods. The value of Re for catalase was taken from Ref. (36) and for the other standard proteins from Ref. (15).

do not dissociate into half-molecules even after incubation for 1 h with 2.2 M NaSCN. The results in Fig. 3 demonstrate that the three bait-region-cleaved complexes, a2M–trypsin, a2M–(succinylated trypsin), and a2M–MeNH2 –SCH3 –trypsin, are dissociated into halfmolecules by 1% SDS but not by 1.6 M NaSCN. Figure 4 demonstrates that the relative fluorescence increases when a2M is incubated for 2 h with 1.6 M NaSCN. The slight red shift in the maximum of the emission spectra corresponding to a 2-h incubation with the salt, probably indicates that the environment of the fluorophores involved is slightly less apolar in

a2M half-molecules than in a2M. The higher values of the relative fluorescence obtained when lex Å 275 nm than those when lex Å 289 nm suggest that energy transfer from tyrosine to tryptophan in the protein may be involved. The curves in Fig. 5 depict the rate of change in relative fluorescence at 340 nm when lex Å 275 and 289 nm, respectively. The curves show that 90% of the final change in fluorescence (DFmax) is reached after about 70 min at lex Å 275 nm and more than 200 min at lex Å 289 nm after the start of incubation of a2M with 1.6 M NaSCN. The first order rate constant, k, calculated from the data in Fig. 5 is 6.1 { 0.6 1 1004 s01 when lex Å 275 nm and 2.1 { 0.2 1 1004 s01 when lex Å 289 nm. The value of k is thus almost threefold higher when the fluorescence is excited at 275 nm than when it is excited at 289 nm. DISCUSSION

The present results clearly demonstrate that NaSCN dissociates a2M into half-molecules with an electrophoretic mobility similar to that obtained upon incubating the protein with 1% SDS (33). The Re of the generated a2M half-molecule is similar to that obtained by dissociation of a2M in 4 M urea (15, 18). Because neither urea nor NaSCN cleave disulfide bonds, the a2M half-molecules described here should be generated by the disruption of the noncovalent interactions between the disulfide-bonded dimers of a2M. Furthermore, it is interesting that the Re of the a2M half-molecule is identical to that of PZP which is a dimer in the native state. Because thiol groups are nearly stoichiometrically exposed by treatment of a2M or a2M half-molecules with methylamine or trypsin, the thiol esters of a2M halfmolecules should be intact. An important result of the present work is that the noncovalent interactions between the disulfidebonded dimers of a2M are sensitive to the conformation of the protein (Table III). Thus native a2M and

TABLE II

The Stokes Radii, Re , of a2M, a2M Half-Molecules, and PZP

Protein

Dissociation conditions

Technique employed for determination of Re

Re (nm)

a2M a2M a2M

None None None

Chromatography Chromatography Ultracentrifugation

9.4 8.8 9.4

a2M a2M a2M a2M a2M PZP

1.6 M NaSCN 0.5 mM DTT 0.5 mM DTT 4 M urea 4 M urea None

Chromatography Chromatography Ultracentrifugation Chromatography Ultracentrifugation Chromatography

7.0 5.7 6.0 7.5 7.7 7.0

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Source This study Roche et al. (13) Roche et al. (13); McConnel and Loeb (16) This study Roche et al. (13) Roche et al. (13) Roche et al. (13) McConnel and Loeb (16) This study

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DISSOCIATION OF a2-MACROGLOBULIN BY SODIUM THIOCYANATE TABLE III

The Degree of Dissociation of a2M and Its Derivatives (as Percentage of Total Protein) into Half-Molecules by Incubation with Different Concentrations of NaSCN for 1 h Concentration of NaSCN (M) Protein

1.2

1.4

1.6

1.8

2.0

2.2

a2Ma a2M–MeNH2 a2M–MeNH2 –CH3 a2M–MeNH2-SCH3 –trypsin a2M–trypsin

12 4 11 0 0

49 26 38 0 0

87 39 73 0 0

91 55 85 0 0

94 83 88 0 0

ndb 87 ndb 0 0

a b

Values for a2M are those obtained by incubation for 30 min. Not determined.

the derivative a2M – MeNH2 – SCH3 , which have earlier been demonstrated to have a similar open-trap structure (30, 34), dissociate to a similar extent when exposed to the same concentration of NaSCN; i.e., they display a similar ‘‘dissociation profile.’’ However, the noncovalent interactions between the disulfide-bonded dimers appear to be strengthened by the conformational changes which yield a closed-trap state of a2M, upon cleavage of the thiol ester, without simultaneous modification of the exposed thiol group, as illustrated by the effect on a 2M – methylamine. These results are in line with earlier observations that the noncovalent interactions between subunits are stabilized by modification of a2M with methylamine (14, 15). The noncovalent interactions appear to be further strengthened when both the thiol esters and bait regions are cleaved, as in the derivatives a2M – trypsin, a2M – (succinylated trypsin), and a2M – MeNH2 – SCH3 – trypsin (Fig. 3). The cleavage in the bait region seems to strengthen the interactions at the dimer – dimer contact surface to

FIG. 3. Electrophoretic analysis of the effect of incubation baitregion-cleaved derivatives of a2M with 1.6 M NaSCN and 1% SDS, respectively. The derivatives used are: a, a2M–trypsin; b, a2M–(succinylated trypsin) and c, a2M–MeNH2 –SCH3 –trypsin. They were prepared by incubating a2M with trypsin or succinylated trypsin, in the molar ratio 1:2.5. Lanes are: 1, control (no incubation); 2, incubation with 1.6 M NaSCN for 1 h at 227C; and 3, incubation with 1% SDS for 90 min at 227C. The electrophoresis was performed under nondenaturing conditions on a 4–15% gradient gel as described under Materials and Methods.

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such an extent that even 2.2 M NaSCN fails to disrupt them (Table III). The difference in the effect of NaSCN on a2M – methylamine and those on the derivatives formed by treatment with trypsin or succinylated trypsin ( a2M – trypsin, a2M – (succinylated trypsin), and a2M – MeNH2 – SCH3 – trypsin), also confirm our earlier observation (10) that there is a difference in the conformation of a2M induced by cleavage of the thiol esters alone and the one induced by simultaneous cleavage of the thiol esters and in the bait regions. Furthermore, as pointed out previously (30), trypsin in a2M – MeNH2 – SCH3 – trypsin is not covalently

FIG. 4. The intrinsic fluorescence of a2M on incubation with 1.6 M NaSCN at 207C. The wavelength of excitation, lex , was at 275 and 289 nm, respectively, and the emission spectra between 320 and 360 nm were collected at zero time and after 120 min, with the time of mixing the solutions of a2M and NaSCN taken as zero. The concentration of a2M in the cuvette was 0.05 mg/ml (when lex Å 275 nm) and 0.1 mg/ml (when lex Å 289 nm), respectively. The slit width was 3 and 5 mm for the excitation and emission beam, respectively. The spectra are: h, j, for lex Å 275 nm; and s, l, for lex Å 289 nm. The unfilled symbols represent spectra at t Å 0 and the filled symbols the spectra at t Å 120 min.

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derstanding of the assembly of this a-macroglobulin and its function. ACKNOWLEDGMENTS Thanks are due to Luis F. Arbelaez for the gift of PZP. The authors also thank Eva-Maj Ha¨gglo¨f for excellent technical assistance. This work has been supported by grants from Umea˚ University (to P.E.H.J.) and Magnus Bergvalls Stiftelse (to V.P.S.).

REFERENCES

FIG. 5. The rate of change in the relative fluorescence (DF) of a2M at 340 nm on incubation with 1.6 M NaSCN at 207C from the type of data depicted in Fig. 4. The data are: s for lex Å 275 nm; and l for lex Å 289 nm. The curves are theoretical curves calculated for a first-order process using the GraFit program.

bound. Therefore, the observed difference in the dissociation profile between this derivative and a2M – MeNH2 – SCH3 must depend solely on the conformational changes induced by cleavage in the bait regions by trypsin. This is supported by the results obtained with the derivative a2M – (succinylated trypsin) (Fig. 3). Succinylated trypsin cleaves in the bait regions of a2M, but is not covalently bound. The difference in conformation of the native a2M and a2M half-molecules is confirmed by the results depicted in Figs. 4 and 5, which clearly show that the dissociation of the tetramer into half-molecules causes an increase in the intrinsic fluorescence and the increase is higher when the fluorescence is excited at 275 nm than at 289 nm. Further studies are necessary to explore why the rate of change in fluorescence is approximately threefold lower when only tryptophan is excited in comparison to when both tryptophan and tyrosine are excited and second why the fluorescence change requires more than 3 h to approach its final value when the fluorescence is excited at 289 nm, while the dissociation, as judged from electrophoresis, takes only 1 h. The high concentration of the chaotropic anion thiocyanate needed to disrupt the noncovalent dimer–dimer interactions suggests the involvement of hydrophobic as well as ionic forces between the noncovalently bonded subunits in a2M. The hydrophobic nature of the interactions between disulfide-bonded dimers might be the reason for the observed increase in hydrophobicity of a2M half-molecules as compared to that of a2M (35). A study of different factors which affect the dissociation of a2M and its complexes would yield a better un-

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1. Sottrup-Jensen, L. (1989) J. Biol. Chem. 264, 11539–11543. 2. Jensen, P. E. H., and Sottrup-Jensen, L. (1986) J. Biol. Chem. 261, 15863–15869. 3. Sottrup-Jensen, L. (1987) in The Plasma Proteins (Putnam, F. W., Ed.), Vol. 5, pp. 191–291, Academic Press, San Diego, CA. 4. Pochon, F., Amand, B., Lavalette, D., and Bieth, J. (1978) J. Biol. Chem. 253, 7496–7499. 5. Salvesen, G. S., and Barrett, A. J. (1980) Biochem. J. 187, 695– 701. 6. Van Leuven, F., Cassiman, J.-J., and Van den Berghe, H. (1979) J. Biol. Chem. 254, 5155–5160. 7. Gonias, S. L., Reynolds, J. A., and Pizzo, S. V. (1982) Biochim. Biophys. Acta 705, 306–314. 8. Jensen, P. E. H., and Stigbrand, T. (1992) Eur. J. Biochem. 210, 1071–1077. 9. Jensen, P. E. H. (1993) in Mechanisms of Proteinase Inhibition by a2-Macroglobulin and Pregnancy Zone Protein, Umea˚ University Medical dissertation, Umea˚, Sweden. 10. Jensen, P. E. H., Ha¨gglo¨f, E. M., Arbelaez, L. F., Stigbrand, T., and Shanbhag, V. P. (1993) Biochim. Biophys. Acta 1164, 152– 158. 11. Arbelaez, L. F., Jensen, P. E. H., Shanbhag, V. P., and Stigbrand, T. (1993) Eur. J. Biochem. 218, 651–666. 12. Feldman, R. S., Gonias, S. L., and Pizzo, S. V. (1985) Proc. Natl. Acad. Sci. USA 82, 5700–5704. 13. Liu, D., Feinman, R. D., and Wang, D. (1987) Biochemistry 26, 5221–5226. 14. Gonias, S. L., and Pizzo, S. V. (1983) Biochemistry 22, 536–546. 15. Roche, P. A., Salvesen, G. S., and Pizzo, S. V. (1988) Biochemistry 27, 7876–7881. 16. Sjo¨berg, B., Pap, S., and Kjems, J. K. (1985) Eur. Biophys. J. 13, 25–30. 17. Barrett, A. J., Brown, M., and Sayers, C. (1979) Biochem. J. 181, 401–408. 18. McConnel, D. J., and Loeb, J. N. (1974) Proc. Soc. Exp. Biol. Med. 147, 891–896. 19. Sottrup-Jensen, L., Petersen, T. E., and Magnusson, S. (1980) FEBS Lett. 121, 275–279. 20. Sjo¨berg, B., Ja¨rnberg, S.-E., and Mortensen, K. (1991) Biochem. J. 278, 325–328. 21. Sjo¨berg, B., Pap, S., and Kjems, J. K. (1987) Eur. J. Biochem. 162, 259–264. 22. Pap, S., Sjo¨berg, B., and Mortensen, K. (1990) Eur. J. Biochem. 191, 41–45. 23. Pochon, F., Barray, M., and Delain, E. (1987) Biochem. Biophys. Res. Commun. 149, 488–492. 24. Eisenstein, E., and Sachman, H. K. (1989) in Protein Function, a Practical Approach (Creighton, T., Ed.), pp. 135–176, IRL Press, Oxford. 25. Chase, T., and Shaw, E. (1970) Methods Enzymol. 19, 20–27.

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