sodium-cyanoborohydride and sodium-borohydride

Previous studies have shown that ethanol pretreatment of glutaraldehyde (GA)-fixed porcine aortic valve cusps (GPAV) significantly reduces bioprosthetic leaflet calcification. The anti-calcification mechanism is due to extraction of cholesterol and phospholipids, and a permanent alteration in collagen structure. It was noted in experimental implants that ethanol-pretreated GPAV occasionally show low levels of calcification. The study aim was to investigate whether this was due to unreacted aldehyde residues and other reducible compounds resulting from GA cross-linking.. GPAV were cross-linked in GA (0.6%) and stored at pH 7.4 in 0.2% GA. Cusps were pretreated with ethanol (80%, pH 7.4) for 24 h. Experimental groups included ethanol-pretreated cusps and GA-fixed controls that were pretreated with either sodium borohydride or sodium cyanoborohydride. Differential scanning calorimetry was used to measure shrink temperature as a measure of cross-linking. Subdermal implants of valve cusp tissue were carried out in 21-day-old Sprague-Dawley male rats. Implants were retrieved at 21 days and samples assessed for the extent of calcification using chemical analyses for Ca, and microscopy studies.. Ethanol pretreatment significantly inhibited calcification compared with controls (13.3 +/- 5.6 versus 119.2 +/- 6.6 micrograms Ca/mg tissue; p < 0.001). However, sodium borohydride reduction under optimized conditions combined with ethanol pretreatment optimally reduced calcification (1.16 +/- 0.1 microgram Ca/mg; p < 0.05), whereas levels after sodium cyanoborohydride treatment (23.6 +/- 10.4 micrograms Ca/mg) were not significantly different to those after ethanol alone. Neither reducing agent was effective in inhibiting calcification without ethanol pretreatment. Furthermore, the reducing agents had no significant effect on shrink temperature.. Inhibition of GPAV calcification with ethanol pretreatment can be enhanced through the optimized use of reducing agents. This indicates that reducible aldehyde-related moieties are likely responsible for breakthrough calcification, even after ethanol pretreatment.

Topics: Animals; Aortic Valve; Bioprosthesis; Borohydrides; Calcinosis; Cross-Linking Reagents; Ethanol; Glutaral; Heart Valve Prosthesis; Male; Rats; Rats, Sprague-Dawley; Reducing Agents; Skin; Solvents

The mutY gene product of Escherichia coli is a 39-kDa protein that catalyzes the removal of adenine bases mispaired with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. Although adenine removal proceeds via monofunctional glycosylase activity, MutY is able to form covalent adducts with substrate DNA in the presence of borohydride, a trait otherwise known to be associated only with enzymes having bifunctional glycosylase/AP lyase activity. To help identify active site residues involved in the formation of MutY-DNA adducts in the presence of borohydride, a series of site-directed mutant forms of MutY were generated. Our data show that Lys 142 is the primary residue involved in cross-link formation. The absence of Lys 142 results in near elimination of the enzyme-DNA adducts formed relative to wild-type, suggesting that this residue is the primary one involved in forming covalent associations with DNA during MutY catalysis. Importantly, the enzymatic activity and DNA binding of the K142A enzyme is nearly identical to the WT enzyme. This shows that formation of the covalent intermediate is not required for adenine removal by MutY. Furthermore, this suggests that the covalent intermediate is formed by reaction of Lys 142 with the OG/G:(AP site) product, and this may be a consequence of MutY's unusually high affinity for the product of its glycosylase action.

Topics: Borohydrides; DNA Adducts; DNA Glycosylases; DNA Repair; Enzyme Activation; Escherichia coli; Lysine; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases; Rifampin; Schiff Bases; Substrate Specificity

Topics: Borohydrides; Gold Colloid; Microscopy, Electron

Sodium borohydride and sodium cyanoborohydride were assessed as potential reagents for determining ligand-induced changes in accessibility to the active-site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determined in the presence of increasing concentrations of the dicarboxylate substrate analogues glutarate and maleate. The rate of reduction decreased to a limiting value which was about 40-fold lower than the equivalent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompanied binding of dicarboxylates. Allowing for this increase, the true decrease in accessibility to NaBH3CN was estimated to be approximately 400-fold. Arguments are presented in support of a proposal that the ratio of closed to open conformer of the dicarboxylate-liganded enzyme is approximately 150. The effects of increasing ligand concentration on the reactivity of Cys390 were found to take place in the same range as was observed for NaBH3CN reduction. Conversely, very much higher concentrations of the dicarboxylates were required to protect against proteolysis by trypsin. It is concluded that NaBH3CN reduction and reactivity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an additional binding mode for the dicarboxylates.

Topics: Animals; Aspartate Aminotransferases; Borohydrides; Cysteine; Glutarates; Hydrolysis; Kinetics; Ligands; Maleates; Molecular Probes; Myocardium; Oxidation-Reduction; Protein Conformation; Swine

Pyridoxylated hemoglobin derivatives have been studied by many investigators. In this study hemoglobin A0 rather than stroma-free hemoglobin was used as a starting material in order to reduce the number of proteins to A0 and A1c. Derivatives were characterized using a Synchropak Q300 strong anion-exchange column, a PolyCAT A weak cation-exchange column and a VYDAC reversed-phase high-performance liquid chromatographic column. Resulting peak profiles of these two ion-exchange separations demonstrated enhanced resolution and showed the presence of pyridoxylated hemoglobin products not previously described. We compared products from the reduction of these Schiff base derivatives using either sodium borohydride or sodium cyanoborohydride reduction procedures. The sodium cyanoborohydride reduction method produced a lower percentage of products with multiple-site pyridoxylation modifications than the sodium borohydride reduction process.

Topics: Blood Substitutes; Borohydrides; Chromatography, High Pressure Liquid; Hemoglobin A; Humans; Pyridoxal Phosphate; Schiff Bases