Cellular imaging and mitochondria targeted photo-cytotoxicity in visible light by singlet oxygen using a BODIPY-appended oxovanadium(iv) DNA crosslinking agent

Targeted PDT by1O2at mtDNA crosslinking site of a BODIPY-appended VO2+complex in visible light.

Why α2-Antiplasmin Must Be Converted to a Derivative Form.

Human α2-antiplasmin (α2AP), the primary inhibitor of plasmin, is secreted from liver to plasma as a 464-residue protein with Met as the N-terminus (Met-α2AP). As it circulates, antiplasmin-cleaving enzyme, a possible derivative of fibroblast activation protein, cleaves the Pro12-Asn13 bond of Met-α2AP to yield Asn-α2AP. The Asn-α2AP form becomes crosslinked to fibrin by activated factor XIII (FXIIIa) ~13X faster than Met-α2AP, and consequently, fibrin clot stability increases in direct proportion to the ratio of Asn-α2AP/Met-α2AP in plasma. FXIIIa, a transglutaminase catalyzes isopeptide bond crosslinking between a primary amine donor Lys in fibrin and an acceptor Gln in α2AP. Using recombinant Asn-α2AP mutants, we reported multiple FXIIIa-catalyzed fibrin crosslinking sites in Asn-α2AP: the previously reported Gln14 (Gln2 in Asn-α2AP) site and our discovery of three other Gln residues. This current study was performed to determine whether the major crosslinking site in Met-α2AP is also Gln14 or whether steric hindrance shifts the predominant crosslinking site to one of the other three identified Gln residues. Native Asn-α2AP and two different forms of Met-α2AP defined by an Arg6Trp polymorphism were labeled with 5-(biotinamido)pentylamine (BPA) by FXIIIa catalysis. To identify FXIIIa-reactive sites in Met-α2AP(Arg6), Met-α2AP(Trp6) and Asn-α2AP, each BPA-labeled α2AP was reduced, alkylated, and digested by trypsin. The BPA-labeled peptides were isolated from the tryptic digest mixture by avidin affinity chromatography, and further separated by reverse-phase HPLC. Using N-terminal sequence analysis of BPA-labeled peptides, we identified only the Gln14 residue as a FXIIIa-reactive site in all three forms of α2AP. Although four Gln sites in recombinant Asn-α2AP were involved in crosslinking, the Gln14 site appears to function as the only significant crosslinking site in the native form of the protein, whether either polymorphic form of precursive Met-α2AP, or the derivative, Asn-α2AP. The finding of Gln14 as the significant crosslinking site in native Asn-α2AP, as opposed to the four sites in recombinant Asn-α2AP, is likely related to differences in glycosylation, since the native protein is glycosylated while the recombinant protein is not. Using liquid chromatography/mass spectrometry analysis of tryptic digested BPA-labeled Asn-α2AP, another FXIIIa-reactive site (Gln33; Gln21 in Asn-α2AP) was identified, but the reactivity of this residue was ~200-fold less than the Gln14 residue. Finally, the BPA-labeling efficiencies of α2AP were determined by avidin blot analysis. BPA was incorporated into all three forms of α2AP by FXIIIa catalysis, but Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA far more slowly than Asn-α2AP, which is consistent with the difference in fibrin-crosslinking rates. Met-α2AP(Arg6) and Met-α2AP(Trp6) became labeled by BPA at a similar rate despite the Arg6Trp polymorphism being located close to the Gln14 used for crosslinking to fibrin. These data suggest that the Gln14 site is cryptic in Met-α2AP, being sheltered by the 12-residue N-terminal peptide, which when cleaved, yields Asn-α2AP that becomes crosslinked to fibrin more quickly to provide greater protection from plasmin.

CONFORMATIONAL EQUILIBRIA IN THE γ CHAIN COOH-TERMINUS OF HUMAN FIBRINOGEN

Synthetic peptides and fragments cleaved from native fibrinogen are used in studies to localize binding sites for various ligands. We addressed the question how the native conformation of a selected γ chain segment is affected by scission of the original chain. The conformation of the γ chain COOH-terminus of intact fibrinogen and its various fragments containing this region has been compared by an immunochemical analysis. An antibody population specific for the native epitope within the γ391-405 segment was isolated by affinity chromatography on the corresponding synthetic peptide. Between 19.2 and 22.8% of antibodies were obtained from three different antisera indicating that this region represents one of the major epitopes of native fibrinogen. Anti-γ391-405 antibodies were used to determine the value of Kconf the equilibrium constant for the interconversion of the non-native and native conformations of this epitope. The measurements were done using native fibrinogen, fragments D1 and DD, γ chain and γ391-405 synthetic peptide. In addition, the effect of 5 M guanidine-HCl on the conformation of fragments D1 and DD, which is known to abolish their antipolymerizing activity, was studied. Radioiodinated fibrinogen was used in the determination of Kconf, and quantitative analytical parameters, CI50% and CIs, calculated from competition between 125I-fibrinogen and the fibrinogen derivatives under study for binding to the immunochemically purified antibody. The measurements indicated that the epitope is unperturbed by iodination of fibrinogen and that 38.3% of fragment D1, 8.9% of fragment DD, 3.6% of the γ chain and less than 0.008% of the γ391-405 molecules adopt in aqueous solution the native conformation within the epitope. Denaturation of fragment D1 with 5 M guanidine-HCl affected only slightly the conformation of this γ chain determinant. More significant changes in the conformation were observed when fragment DD was denatured. The results suggest that long-range interactions are necessary for the stabilization of the native structure in the region of fibrinogen that interacts with the antibody and which is in close vicinity to the polymerization site, crosslinking site, and platelet recognition site.