German B. Villanueva

Evidence for a heparin-induced conformational change on antithrombin III☆

Abstract The effect of heparin on the conformation of antithrombin III (AT-III) was investigated. Solvent perturbation difference spectroscopy shows that the binding of heparin to AT-III results in exposure of two tyrosine residues and a partial burial of a tryptophan residue. The occurrence of a conformational change suggested by this study is also substantiated by circular dichroism (CD) findings in the aromatic and peptide regions. The data in the peptide region show that heparin produces a decrease in the β-structure of AT-III, with a compensatory increase in random coil.

Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin

Highly purified hirudin with a specific activity of 13,950 antithrombin units/mg was isolated from a commercial preparation by reversed-phase chromatography. The circular dichroism (CD) spectrum of hirudin was investigated and it was found that the spectrum cannot be accounted for solely in terms of the traditional three components of peptide backbone. It was also found that the CD spectrum of the thrombin-hirudin complex was not additive with respect to the individual spectra of thrombin and hirudin. This deviation from additivity was significant between 210 and 225 nm, indicating alterations in the secondary structures of the proteins during complex formation. When thrombin was titrated with hirudin, the spectral deviation from additivity was sigmoidal, suggesting the cooperative nature of the binding process. Gel filtration of the thrombin-hirudin mixture showed no molecular species greater than a 1:1 complex (Mr 45,500), but gel filtration of free hirudin showed a multimeric form (Mr 51,300) under the same experimental conditions. It is concluded that the cooperative nature of the binding process is due to the binding of thrombin molecules to the multimeric form of hirudin. This initial binding occurs with little or no change in the CD spectrum. In the second step, the multiple complex dissociates to form 1:1 complexes, resulting in larger conformational changes and a considerable increase in binding affinity.

Demonstration of Altered Antithrombin III Activity Due to Nonenzymatic Glycosylation at Glucose Concentration Expected to be Encountered in Severely Diabetic Patients

The effect of nonenzymatic glycosylation on the kinetics and structure-function relationships of antithrombin III were investigated at normal physiologic concentrations of antithrombin III and glucose, which are 5.2 μM and 5 mM, respectively. The results were compared with antithrombin III incubated at the glucose concentration expected to be found in severely diabetic patients (15 mM). Antithrombin III incubated at 5 mM lost 33% of the heparin cofactor activity after 7 days, whereas antithrombin III incubated at 15 mM lost 50% for the same period. Under both conditions, half of the heparin cofactor activity was lost after 15 days. When D-[U-14C]glucose was used as tracer, ∼0.6 mol glucose/mol protein was incorporated after 10 days at both concentrations of glucose. A detailed evaluation of the kinetics of inhibition of thrombin by glycosylated antithrombin III revealed that the second-order rate constant is three times smaller than that of normal antithrombin III. On the basis of these data, it is concluded that glycosylated antithrombin III with 50% depressed heparin cofactor activity is three times weaker than normal antithrombin III as an inhibitor of thrombin. The implications of these observations with respect to the possible pathogenesis of thrombosis in diabetes are discussed.

Tryptophan residue at the heparin binding site in antithrombin III

Abstract Chemical modifications have demonstrated that the ultraviolet difference spectrum produced when heparin interacts with antithrombin III is due primarily to changes in the tryptophan environment. This is based on the observation that this spectrum could be abolished by treatment of antithrombin III with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide but not with tetranitromethane. The tryptophan-modified antithrombin III is still capable of binding to thrombin even when it has lost 85% of heparin cofactor activity. A marked decrease in reactivity of tryptophan residues is observed when modification is carried out in the presence of heparin. Evidence is presented that tryptophan is in the heparin binding site.

Subunit dissociation of Busycon canaliculatum hemocyanin

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)

Conformation of high molecular weight kininogen: Effects of kallikrein and factor XIa cleavage*

The effect of kallikrein and factor XIa proteolysis of high molecular weight kininogen (HK) was investigated. Circular dichroism (CD) spectroscopy showed that cleavage of HK by plasma kallikrein or urinary kallikrein, both of which result in an active cofactor (HKa), results in conformational change that is characterized by increase in CD ellipticity at 222 nm. This suggests an increase in organized secondary structures. By contrast, cleavage of HK by factor XIa which results in an inactive cofactor (HKi) is characterized by a dramatic decrease in CD ellipticity at 222 nm suggesting an entirely different type of conformational change. The intrinsic fluorescence of HK is enhanced after cleavage by all three proteases. These conformational changes may play a role in determining the structure and function of HKa and HKi.

Structure-function relationships in heparin cofactor II: chemical modification of arginine and tryptophan and demonstration of a two-domain structure

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 M GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.

Effects of sodium and lithium salts on the conformation of human α-thrombin

Abstract Chemical modification studies have demonstrated that the ultra-violet difference spectrum of α-thrombin produced in the presence of sodium is due primarily to changes in the environment of tyrosine residues. This is based on the observation that the spectrum could be abolished by treatment of α-thrombin with tetranitromethane but not with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Although lithium produces similar (UV) difference spectrum, circular dichroism studies indicate that sodium and lithium induce different conformational transitions. α-Thrombin tends to assume a more ordered structure in the presence of sodium whereas lithium has the reverse effect. This inverse hehavior is consistent with the effects of these cations on the autolysis rate and thermal stability of the activities of α-thrombin.

Effect of heparin modification on its circular dichroism spectrum

The effect of modification of the carboxyl groups of high affinity heparin was investigated. The binding affinity toward antithrombin III decreases in the following order: Heparin greater than heparin methyl ester greater than heparinylglycine greater than heparinylglycine methyl ester. This result agrees qualitatively with the previous studies using unfractionated heparin. Esterification of the carboxyl groups (i.e., HME) does not affect the CD profile of heparin at 210 nm but introduction of a bulkier glycine methyl ester (i.e., HGME) leads to formation of a very intense band at 235 nm. Based on reported CD analyses of uronic acid derivatives and our model building studies, it is concluded that the large difference in CD spectra of HGME as compared to unmodified heparin and HME is due to a change in ring conformation of the uronic acid moiety (i.e., 4C1 to 1C4 or vice versa).

Circular dichroism of platelet factor 4

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.