Borohydride Reduction Method Research Articles

Refinement of the Borohydride Reduction Method for Trace Analysis of Dissolved and Particulate Dimethyl Sulfoxide in Marine Water Samples

A recently reported borohydride reduction method for the trace determination of aqueous dimethyl sulfoxide (DMSO) was adapted for use with a different sample preparation and analytical system, and the adaptation and optimization steps that we followed gave us further insight into the method. Increasing the proportion of reducing agent was critical. A number of compounds with potential for analytical interference were tested, but all proved negative. Water blanks were problematic, with substantial DMSO contamination observed in all but very recently purified water. Preliminary comparison with the highly specific and precise enzyme-linked method gave very good agreement. When DMSO analysis was done sequentially after analysis of dimethyl sulfide and alkali hydrolysis for dimethylsulfoniopropionate, we found that the DMSO concentration was not affected by increasing the length of the hydrolysis step. This allows storage and/or transport of hydrolyzed samples in gastight containers. The adapted method was applied to the novel determination of DMSO on glass fiber filters, and this revealed a significant pool of particulate DMSO in marine particles.

A new method of preparation of iron(II) porphyrin complexes—isolation and characterization of amine complexes of ferrous porphyrin

The six-coordinate complexes [Fe II(TTP)(NHR 2) 2] (TPP = meso-tetraphenylporphyrinate, NHR 2 = NH 2CH 2Ph, NHMe 2 have been prepared by the reduction of Fe III porphyrin with amines in sodium hypochlorite solution in air as well as by the sodium borohydride reduction method. The complexes were characterized by UV-vis and IR spectroscopies and elemental analysis.

Intracellular predominance of the pyridoxal 5′-phosphate form of aspartate aminotransferase in Escherichia coli B and reversible transformation of this form by extracellular substances

The intracellular proportion of the pyridoxal 5′-phosphate form of aspartate aminotrasferase to the total enzyme in E. coli B cells was determined by a newly devised method, dependent on selective inactivation of the intracellular pyridoxal 5′-phosphate form of the enzyme by extracellularly added sodium borohydride. A large portion (80–99%) of the intracellular aspartate aminotransferase was in pyridoxal 5′-phosphate form in both natural and synthetic medium-grown bacterial cells. The intracellular predominancy of pyridoxal 5′-phosphate did not vary during the growth of bacteria and during incubation of bacterial cells in various kinds of buffers with different pH values. In contrast, the saturation levels generally used to describe in vivo the proportions of the apo and holo vitamin B 6-dependent enzymes did not reflect the intracellular amount of the pyridoxal 5′-phosphate (holo) form of aspartate aminotransferase probably because the intracellular pyridoxal 5′-phosphate form was changed to an apo form by the disruption of bacterial cells for preparing crude extract. Various extracellularly-added vitamin B 6 antagonists decreased the intracellular amount of pyridoxal 5′-phosphate without decrease in the total intracellular activity of the enzyme. The modified forms were stable in E. coli B cells and reversed into pyridoxal 5′-phosphate form by incubation of the antagonist-treated cells in the buffer containing pyridoxal. The present results showed that the sodium borohydride reduction method can be used for further analysis of the in vivo interaction of pyridoxal 5′-phosphate and apoaspartate aminotransferase. The fact that about 50% of the intracellular pyridoxal 5′-phosphate form was changed to a modified form without impairment of cell growth in the presence of 4-deoxypyridoxine, and that about 50% of intracellular modified asparate aminotransferase was reversed to pyridoxal 5′-phosphate by the removal of antagonist followed by incubation suggested that there exists characteristically 2 different fractions of pyridoxal 5′-phosphate forms of aspartate aminotransferase in E. coli cells.

Defective lysosomal release of glycoprotein-derived sialic acid in fibroblasts from patients with sialic acid storage disease

Fibroblasts from patients with sialic acid storage disease (SASD), sialidosis, mucolipidosis II, and from normal controls, were incubated in the presence of the glycoprotein fetuin that was tritium-labelled in its sialic acid residues by the periodate/[3H]borohydride reduction method, and the fate of the intracellular radioactive sialic acid (C7-sialic acid) followed in pulse-chase experiments. The model glycoprotein was readily endocytosed and degraded, more than 90% of the radioactivity being trichloroacetic acid (TCA)-soluble after 4 days of incubation. In all of the patients' fibroblasts, there was an increased accumulation of TCA-soluble radioactivity and, upon chase, a much lower rate of elimination than in normal controls. Gel chromatography of the material from the chase experiment showed that, in normal cells, most of the radioactivity at zero time behaved as free C7-sialic acid. This, as well as material of larger size (sialyloligosaccharides), was very much diminished by 48 h. In cells from two patients with SASD, there were large peaks both in the sialic acid and oligosaccharide positions; whereas the oligosaccharides were somewhat decreased by the end of the chase period, the sialic acid was essentially unchanged. In sialidosis fibroblasts, the radioactive material consisted of oligosaccharides, but very little C7-sialic acid; the elimination of the oligosaccharides was retarded. In normal cells, about 80% of the radioactivity released into the medium after 48 h chase behaved as free C7-sialic acid upon gel chromatography and t.l.c. Subcellular fractionation in Percoll gradients showed that the radioactive C7-sialic acid remaining in normal cells after 48 h of chase was mainly localized in the cytosol. In SASD cells, on the other hand, it was associated with lysosomal fractions which, unexpectedly, exhibited an abnormally low density. Our findings demonstrate that SASD fibroblasts degrade the sialoglycoprotein but, unlike normal cells, accumulate the liberated C7-sialic acid along with sialyloligosaccharides in their lysosomes. The results therefore support the concept of a defective transport system for sialic acid in the lysosomal membrane of patients with SASD.

Evidence for a β-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO(4) at pH 6.5 and then treated with sodium [(3)H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110 degrees C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [(3)H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the beta-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.

The determination of tellurium by nondispersive atomic fluorescence spectrometry using the hydride generation technique

Atomic fluorescence spectrometry with a nondispersive measuring system is combined with a hydride generation technique for the determination of tellurium. Atomic fluorescence measurement is based on the reduction of tellurium by either metallic zinc or sodium borohydride, introduction of the generated tellurium hydride into a premixed argon (entrained air)-hydrogen flame, and excitation with a tellurium electrodeless discharge lamp. The comparison of the zinc and the sodium borohydride reduction methods is discussed in terms of detection limit, precision and interference. The best attainable detection limits for tellurium are 2ng (0.1 ng ml ) and 30 ng (1.5 ng ml ) with the zinc and the sodium borohydride methods respectively. Analytical working curves obtained from peak-height and peak-area measurements are linear over a range of approximately 4 orders of magnitude. Of the mineral acids examined in the range up to 2.0 m. nitric acid gives a depressing interference in the range greater than 0.5 m in the zinc method, whereas all of the acids greater than 1.0 m give a slight enhancement of the signal in the sodium borohydride method. The presence of several elements including other hydride-forming elements in 1000-fold ratio to tellurium causes a depressing interference, while enhancing interferences from tungsten and vanadium are observed in the zinc and the sodium borohydride methods, respectively. The present system coupled with the zinc method has been applied to the determination of tellurium in several samples of high-purity copper metal after separation of the analyte from copper by passing an ammoniacal solution of the sample through Chelex-100 resin. The results are in good agreement with those obtained by graphite furnace atomic absorption spectrometry.

The Determination of Trace Amounts of Selenium by Hydride Generation-Nondispersive Flame Atomic Fluorescence Spectrometry

A method has been developed for the determination of trace selenium by generation of its gaseous hydride using either metallic zinc or sodium borohydride as a reductant and followed by the introduction of the hydride into a premixed argon (entrained air)-hydrogen flame where all atomic fluorescence lines of selenium are simultaneously detected by use of a nondispersive system. The comparison of the zinc and the sodium borohydride reduction methods is discussed in terms of detection limit, precision, and interference. The best attainable detection limits for selenium are 0.3 ng (15 pg/ml) and 0.4 ng (20 pg/ml) with the zinc and sodium borohydride methods, respectively. Analytical calibration curves obtained by measuring peak heights and integrated peak areas of the fluorescence signals are linear over the range of approximately 4 orders of magnitude from the detection limits. Most mineral acids examined in the range up to 2.0 M do not interfere; however, the nitric acid is an exception which gives a depressing interference. The interference was found to be much more severe in the zinc method than in the sodium borohydride technique. The presence of several elements including other hydride-forming elements in 1000-fold ratio to selenium causes a depressing interference, whereas an enhancing interference from tellurium is observed only in the zinc reduction method. The present method has been successfully applied to the determination of selenium in wastewaters and foods.

A Borohydride Reduction Method for Characterization of the Acyl Phosphate Linkage in Proteins and Its Application to Sarcoplasmic Reticulum Adenosine Triphosphatase

A new method for identification and characterization of an acyl phosphate linkage in phosphorylated proteins is presented. The method involves reductive cleavage of the acyl phosphate bond with sodium [3H]borohydride to form a labeled aminohydroxy acid residue. [3H]Borohydride reduction of the phosphorylated (Ca2+, Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum, followed by analysis of the acid hydrolysate of the reduced enzyme, showed the formation of labeled homoserine. The results demonstrate that the phosphoryl group of sarcoplasmic reticulum ATPase is attached to the β-carboxyl group of an aspartyl residue at the active site.

Determination of Carbonyl Group in Cellulose by Sodium Borohydride Reduction Method

There are two methods for the determination of carbonyl group in cellulose by means of reduction with NaBH4. One is the gasometric method proposed by Lindberg et al., and another is the volumetric method proposed by Lyttle et al. The former involves some troubles in its analytical operation. Therefore the authors adopted the latter, and carried out some experiments in order to check up the method.An analytical method now established is as follows. After a sample containing carbonyl group, such as D-glucose or oxydized cellulose by HIO4, is immersed in 0.010.13 M NaBH4 (pH 12.012.3) for 2448 hrs at room temperature and then filtered, a 0.040.4 N KIO3 solution and a 0.5 N NaOH solution are added to the filtrate successively. The amounts of consumed NaBH4 are calculated from the amounts of consumed KIO3 estimated by a titration With a 0.1 N Na2S2O3 solution in acidic condition.At a range of pH 12.012.3, 57% of the amount of consumed NaBH4 are effective for the reduction of carbonyl group in the sample. This is confirmed by the amounts of non-reacting hydrogen gas estimated with a gas burett.The results of treatment of D-glucuronolaeton and the oxydized cellulose by NaBH4 at pH 12.012.3 show that the carboxyl group and its lacton are resistant to the reduction.