Human antiplasmin, a fast-acting inhibitor of plasmin in plasma, belongs to the serpin super-family of proteins. Like other members of this family, antiplasmin has a scissile peptide bond exposed within a reactive centre loop, typically present at the surface of the molecule. Antiplasmin is stable at neutral pH, but at acidic pH or at elevated temperatures it rapidly becomes inactivated. Data regarding “native” antiplasmin have demonstrated that both polymerization processes and formation of latent molecules are important in this respect. In this work we used site-directed mutagenesis to produce 11 single-site mutants (mainly within Aβ-sheet, Bβ-sheet and reactive centre loop), which were expressed in Drosophila S2 cells, purified and characterized. Five of the 11 mutants were found to have a deviating stability at decreased pH. Glu346Thr was the only mutant with a lesser stability as compared to wt-antiplasmin, but the other 4 were more stable. The most stable mutant, His341Thr, was 7-fold more stable at pH 4.9 as compared to wt-antiplasmin. The wt-antiplasmin had a much more pronounced tendency to polymerize at decreased pH, as compared to “native” antiplasmin. However, many of the mutants clearly rather formed latent molecules, as judged both from PAGE-analysis at non-denaturing condition and reactivation experiments.

Several serine proteinase inhibitors (serpins) are metastable proteins which under certain conditions may undergo conformational changes resulting in the insertion of the reactive centre loop into the so-called Aβ-sheet and hence forming latent molecules. Here we have studied the inactivation of antiplasmin as a function of pH and temperature with time. At decreased pH (4.9–5.8) and at room temperature, antiplasmin activity decreased following first-order kinetics. Analysis by polyacrylamide gel electrophoresis under non-denaturing conditions demonstrated that only minor amounts of polymerized material formed after extensive incubation (4 days) at room temperature. However, on incubation at elevated temperatures (45 or 55 °C), a rapid formation of polymerized material was observed. We also demonstrated that antiplasmin inactivated by treatment at pH ∼5 at room temperature spontaneously slowly regained some activity if incubated in a buffer of neutral pH. Furthermore, by treatment with 4 M guanidinium chloride for about 30 min, followed by dialysis against a neutral phosphate buffer, considerable activity (almost 40%) was regained. Thus, we conclude that antiplasmin, at least partially, at lower temperatures is transformed into a latent form, which could be reactivated, in a similar manner as PAI-1. At increased temperature, however, polymerization seems to be the predominant reason for inactivation.

The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.

The kinetics of the urea-induced dissociation of human plasma α2-macrogrobulin to half-molecules has been studied as a function of temperature by using small-angle scattering of X-rays and neutrons. The most striking result of the present investigation is that there is a minimum in reaction rate at about 15 °C, and that the rate increases when the temperature is lowered, or raised, from that value. By analyzing the first-order rate constants in terms of transition-state theory it was found that the dissociation is associated with a large and positive change in heat capacity between the activated complex and native α2-macroglobulin (ΔCP‡, is in the range 5 to 6 kJ mol−1 K−1). In analogy with pure thermodynamic investigations, where a large change in heat capacity normally is interpreted as a melting of hydrophobic interaction, we therefore propose that hydrophobic interaction is involved in the so-called non-covalent interactions between the subunits of α2-macroglobulin. As a result of the present investigation, it also follows that the free energy of activation ΔG‡ has a maximum at about 32 °C, whereas the enthalpy of activation ΔH‡ and the entropy of activation ΔS‡ are zero at about 15 °C and 32 °C, respectively. These temperatures are slightly dependent upon the concentration of urea and upon whether the reaction is run in a 1H or a 2H medium. Furthermore, from the kinetic point of view, at low temperature the reaction can be characterized as enthalpy driven, whereas at high temperature, it can be characterized as entropy driven.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2-macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein.

The dissociation of the tetrameric alpha 2-macroglobulin molecule into two half-molecular fragments, which occurs at pH less than 4.5, has been investigated using the small-angle neutron scattering method, and test of trypsin binding activity. Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid-base groups, one per monomeric subunit of alpha 2-macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H(+)-binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2-4.5, suggests that they are most probably carboxylate groups. From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein. Thus, at pH less than 4.5, there is a transition of alpha 2-macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.

The molecular organization of human plasma alpha 2-macroglobulin (alpha 2M), and its 1:1 and 1:2 trypsin complexes, have been investigated using the small-angle x-ray scattering method. All the experimental data can be explained by the same basic model, consisting of three oblate-shaped domains arranged in a sandwich-like structure. Each of the larger peripheral domains consists of two parallel elliptic cylinders associated side-by-side, whereas the smaller central domain consists of just one elliptic cylinder. In the native molecule the three domains are separated by regions of low protein density. Upon trypsin binding the dimensions of the four peripheral cylinders remain unchanged, but their positioning in space is reorganized so that the whole molecule becomes more compact. The model thus offers a plausible explanation for the mechanism of inactivating of the protease by entrapping it between the two larger domains. By comparing the shape and dimensions of the total molecule with those determined for the half-molecular fragment, obtained after reducing the intersubunit disulfide bonds, we propose that the fragment consists of just one of the peripheral domains plus half of the central domain. Different projections of the model are consistent with the electron micrographs of alpha 2M given in the literature. The model can also explain many of the physical and chemical properties recorded for alpha 2M and its protease complexes.

The structure of human plasma fibronectin in 50 mM Tris-HCl buffer, pH 7.4, containing varying concentrations of NaCl, has been investigated using the small-angle X-ray method.

The dodecylsulfate-induced dissociation of the tetrameric alpha 2-macroglobulin molecule from human plasma has been investigated by the small-angle neutron scattering (SANS) method. The great advantage with the SANS method is that, by using deuterated dodecylsulfate, and contrast variation by changing the D2O/H2O ratio of the solvent, we can selectively study just the protein part, or the dodecylsulfate part, of the protein-dodecylsulfate complex. More than a thousandfold excess of dodecylsulfate (on a molar basis) is needed in order to dissociate alpha 2-macroglobulin to particles with, on average, half the original molecular mass. By combining the SANS data with results obtained by the equilibrium dialysis technique it follows that, under these circumstances, approximately one thousand dodecylsulfate molecules are associated per alpha 2-macroglobulin molecule. From the significant increase in the radius of gyration, which accompanies the dissociation process, we can conclude that the dissociation is associated with a drastic change in conformation of the protein molecule. From measurements where the dodecylsulfate part of the complex dominates the SANS signal we also get an indication that the dodecylsulfate is randomly distributed along the polypeptide chain, rather than being arranged in large clusters at certain regions of the protein molecule. By fitting the parameters of a binding model to the experimental data we obtain the result that most of the more than one thousand bound dodecylsulfate molecules, necessary for dissociation, are involved in the change in conformation, and the dissociation process is, in fact, driven by the binding of a very few extra dodecylsulfate molecules to the dissociation products. These data indicate that the dodecylsulfate-induced dissociation of alpha 2-macroglobulin is probably more complicated than just breaking, for instance, a hydrophobic interaction.

Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly larger values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H2O/g protein (dry weight). Neutron data, recorded at different D2O concentrations in the solvent, gave a match point of 43% D2O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.

The effect of temperature on human α2-macroglobulin dissolved in a water buffer containing 0.0065 m NaH2PO4 and 0.0652 m Na2HPO4 pH 7.78, has been investigated in the range −15°C (supercooled water solutions) to +37°C using small-angle X-ray scattering and dilatometry. From the small-angle X-ray data it follows that there is no detectable change in conformation, or outer dimensions, of α2-macroglobulin as a function of temperature. There is, however, a marked increase in the scattering power of the samples at decreasing temperature and vice versa. From measurements of the influence of the temperature on the apparent specific volume, using dilatometry, it follows that dv1/dt = 0.00055 cm3g−1K−1, and the influence of the temperature on the scattering power can be fully explained by this value. In order to explain this obvious paradox, that there is a change in the apparent specific volume of α2-macroglobulin without any accompanying change in the outer dimensions, we propose that there is also a change in the internal hydration of the protein as a function of temperature. Decreasing temperature leads to an increased hydration and vice versa.

A half-molecular fragment of α2-macroglobulin has been prepared by reducing and alkylating the inter-subunit disulfide bonds in the tetrameric α2-macroglobulin molecule with 1 mM dithiothreitol (40 min) and 3 mM iodoacetamide (40 min). Further purification was made by gel chromatography and the homogeneous population of halfmolecules has been characterized by the techniques of small-angle X-ray and neutron scattering. The radii of gyration found by the two methods are 57.0 and 58.0 Å, respectively. The match point, obtained by neutron scattering from solutions with different H2O/D2O rations, is at 43% D2O; the data are consistent with a particle having a higher scattering density at large distances from the particle centre. From the X-ray and neutron intensities scattered at zero angle, the specific volume was determined to be 0.73 cm3/g at+5°C and the molecular weight to be 390,000; the latter value is associated with a relatively large error due to the uncertainty in the concentration determination. Shape analysis indicates that the best-fitting scattering-equivalent threeaxial bodies are oblate shaped, with two of their axial dimensions about three to four times larger than the third one. From the volume of the best-fitting scattering-equivalent three-axial bodies, 0.72×106 Å3, we obtain a water content equal to 0.38 g H2O/g protein (dry weight).

X-Ray scattering study of alpha 2-macroglobulin in solvents of variable electron densities (sucrose in water) shows that alpha 2-macroglobulin obeys the invariant volume hypothesis; thus, the structure of the particle is independent of the sucrose concentration of the solution. The particle structure is quantitatively described by a set of parameters such as the gyration radius, R = 8.0 nm, the volume, V = 1200 nm3, and the maximum distance within the particle, Dmax = 25 nm. The contrast dependence of the gyration radius indicates that in alpha 2-macroglobulin the regions of higher electron density are located closer to the center (core) than the regions of lower electron density. The core, which may be the place occupied by the carbohydrate, has a maximum dimension of 16 nm and it can be described as a flat cylinder. X-Ray scattering titrations indicate that alpha 2-macroglobulin forms a 1:2 complex as the main product with both trypsin and chymotrypsin simultaneously as the particle contracts. The formation of a ternary 1:1:1 complex with trypsin and chymotrypsin and the absence of higher complexes indicate that the sites for these proteases are closely related. This is further substantiated by the p(r) functions which are virtually identical for the 1:1:1 and 1:2 complexes.

A homogeneous IgG3 protein and its corresponding Fch and Fc fragments have been studied in solution using the small-angle X-ray scattering method. The Fch and Fc fragments were produced by short digestion of IgG3 with papain and trypsin. The results indicate that the overall shape of the IgG3 molecule in solution can best be described as an elliptical cylinder with a total length of 29 nm and with a cross-section having the semiaxes 3.8 and 0.9 nm. Thus, the overall shape of IgG3 is considerably different from the Y-shape normally adopted for the IgG1 molecule. The analysis of the data obtained for the Fch and Fc fragments also yields elliptical cylinders with almost the same dimensions of the cross-section but with shorter total lengths, 11.4 and 6.7 nm, respectively. The molecular weights of the IgG3 protein and the Fch and Fc fragments were determined to be 1.8 · 105, 0.61 · 105, and 0.50 · 105, respectively.