ABSTRACT
For the first time, by using mass-spectrometry method, the oxidation-mediated modification of the catalytic FXIII-A subunit of plasma fibrin-stabilizing factor, pFXIII, has been studied. The oxidative sites were identified to belong to all structural elements of the catalytic subunit: the ß-sandwich (Tyr104, Tyr117, and Cys153), the catalytic core domain (Met160, Trp165, Met266, Cys328, Asp352, Pro387, Arg409, Cys410, Tyr442, Met475, Met476, Tyr482, and Met500), the ß-barrel 1 (Met596), and the ß-barrel 2 (Met647, Pro676, Trp692, Cys696, and Met710), which correspond to 3.9%, 1.11%, 0.7%, and 3.2%, respectively, of oxidative modifications as compared to the detectable amounts of amino acid residues in each of the structural domains. Lack of information on some parts of the molecule may be associated with the spatial unavailability of residues, complicating analysis of the molecule. The absence of oxidative sites localized within crucial areas of the structural domains may be brought about by both the spatial inaccessibility of the oxidant to amino acid residues in the zymogen and the screening effect of the regulatory FXIII-B subunit.
Subject(s)
Catalytic Domain , Factor XIII/chemistry , Factor XIII/metabolism , Humans , Oxidation-Reduction , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
For the first time, the induced oxidative modification of cellular fibrin-stabilizing factor (cFXIII) has been studied. According to the electrophoresis analysis, the conversion of oxidized cFXIII into FXIIIa resulted in producing the enzyme that significantly lost the initial enzymatic activity. At the same time, FXIIIa subjected to induced oxidation was completely devoid of enzymatic activity. The results of FTIR spectroscopy showed that the oxidation of cFXIII or FXIIIa was accompanied by profound changes both in chemical and spatial structures of the protein. The results of this study are in good agreement with our earlier assumption regarding the antioxidant role of the regulatory subunits B of plasma fibrin-stabilizing factor.
Subject(s)
Factor XIII/chemistry , Calcium/chemistry , Cations, Divalent/chemistry , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Factor XIII/isolation & purification , Female , Humans , Oxidants/chemistry , Oxidation-Reduction , Ozone/chemistry , Placenta/chemistry , Polymers/chemistry , Pregnancy , Protein Conformation , Spectroscopy, Fourier Transform InfraredABSTRACT
The effect on ozone-induced oxidation on the self-assembly of fibrin in the presence of fibrin-stabilizing factor FXIIIa of soluble cross-linked fibrin oligomers was studied in a medium containing moderate urea concentrations. It is established that fibrin oligomers were formed by the protofibrils cross-linked through γ-γ dimers and the fibrils additionally cross-linked by through α-polymers. The oxidation promoted both the accumulation of greater amounts of γ-γ dimers and the formation of protofibrils, fibrils, and their dissociation products emerging with increasing urea concentrations, which have a high molecular weight. It is concluded that the oxidation enhances the axial interactions between D-regions of fibrin molecules.
Subject(s)
Fibrin/chemistry , Protein Multimerization , Factor XIII/chemistry , Factor XIII/metabolism , Fibrin/metabolism , Humans , Oxidation-Reduction , Protein Binding , Urea/chemistryABSTRACT
The crosslinking of fibrin γ-polypeptide chains under the influence of the plasma fibrin-stabilizing factor (FXIIIa), which causes their conversion to γ-γ dimers, is the major enzyme reaction of covalent fibrin stabilization. We studied the self-assembly of soluble cross-linked fibrin oligomers. The results of analytical ultracentrifugation as well as elastic and dynamic light scattering showed that the double-stranded fibrin oligomers formed under the influence of moderate concentrations of urea are cross-linked only due to formation of γ-γ dimers, which can dissociate into single-stranded structure when the concentration of urea increases. This fact proves that γ-γ dimers are formed in the end-to-end manner.
Subject(s)
Fibrin/chemistry , Dimerization , Dynamic Light Scattering , Factor XIIIa/chemistry , Humans , Molecular Weight , Protein Multimerization , Ultracentrifugation , Urea/chemistryABSTRACT
Ozone-induced oxidation of fibrinogen has been investigated. The conversion of oxidized fibrinogen to fibrin catalyzed either by thrombin or by a reptilase-like enzyme, ancistron, in both cases is accompanied by production of gels characterized by a higher weight/length ratio of fibrils in comparison with the native fibrin gels. IR spectra of the D and E fragments isolated from unoxidized and oxidized fibrinogen suggest a noticeable transformation of functional groups by oxidation. A decrease in content of N-H groups in the peptide backbone and in the number of C-H bonds in aromatic structures, as well as a decrease in the intensity of the C-H valence vibrations in aliphatic fragments CH2 and CH3 were found. The appearance in the differential spectra of the D fragments of rather intense peaks in the interval of 1200-800 cm(-1) clearly indicates the interaction of ozone with amino acid residues of methionine, tryptophan, histidine, and phenylalanine. Comparison of the differential spectra for the D and E fragments suggests that fibrinogen fragment D is more sensitive to the oxidant action than fragment E. Using EPR spectroscopy, differences are found in the spectra of spin labels bound with degradation products of oxidized and unoxidized fibrinogen, the D and E fragments, caused by structural and dynamical modifications of the protein molecules in the areas of localization of the spin labels. The relationship between the molecular mechanism of oxidation of fibrinogen and its three-dimensional structure is discussed.
Subject(s)
Fibrinogen/chemistry , Ozone/chemistry , Diffusion , Fibrinogen/isolation & purification , Humans , Molecular Structure , Molecular Weight , Oxidation-Reduction , Ozone/pharmacologyABSTRACT
Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350-450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.
