Sodium borohydride radioactive

Treatment with sodium borohydride of the enzyme-substrate complex of aldolase A and dihydroxyacetone phosphate leads to formation of a covalent linkage between the protein and substrate. This and other evidence suggested a Schiff base intermediate (Eq. 13-36). When 14C-containing substrate was used, the borohydride reduction (Eq. 3-34) labeled a lysine side chain in the active site. The radioactive label was followed through the sequence determination and was found on Lys 229 in the chain of 363 amino acids.186/188 188b Tire enzyme is another (a / P)8-barrel protein and the side chain of Lys 229 projects into the interior of the barrel which opens at the C-terminal ends of the strands. The conjugate base form of another lysine,... 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

On reduction with sodium borohydride, product II yielded a pair of radioactive products that were tentatively identified as 2-deoxy-L-Zyxo-hexose and 2,6-dideoxy-L-arafeino-hexose. This result, together with the chromatographic properties of II, and its reaction on chromatograms with vanillin-perchloric acid to give a blue color, are consistent with the structure of 2,6-dideoxy-L-Zyxo-hexos-4-ulose (20) for II. This structure is also consistent with the concept that II (20) is, very probably, the precursor of III (19). 

Radioactive [35S]-phosphorothioates have been used for in vivo evaluation of chronic or cumulative toxicity of oligonucleotides. To achieve site-specific 35S-labeling in phosphoramidite chemistry, either a mixture of [35S]/carbon disulfide/pyridine/triethylamine [329] or 35S-la-beled Beaucage reagent [330] have been used in the sulfurization step. 3H- and 14C-labeled compounds have been tools in investigations of the metabolic fate of the nucleobases of an oligomer [331]. 3H may be inserted at the 5 -end by formation of the 5 -carboxyaldehyde by Mofatt-Pfitzner reagent, followed by reduction with [3H]-sodium borohydride in 2-propanol... 

The absolute stereochemistry of ipecoside was determined by correlation studies with dihydroprotoemetine. Because the yield of the product in each step was low and a complex mixture of compounds was formed during the reaction, recourse was taken to the use of radioactive material. Vigorous acid hydrolysis of [0-methyl-3H]0,0-dimethyldihydroipecoside furnished 15 or 16 in equilibrium with 17 or 18, and also several isomeric benzo-quinolizidines. From this, after sodium borohydride reduction, (— )-[0-methyl-3H]dihydroprotoemetine (19) of known absolute configuration... 

For the analysis of complex oligosaccharides which can often be present in quite low concentrations, radioactive labelling with tritiated sodium borohydride has been used and allows more accurate and sensitive quantification (Warner et al., 1983 Mellis and Baenziger, 1983). 

An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-OP) The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. The [ S] MMPR-OP can be prepared easily for radioactive affinity labeling of the enzyme. 

The Schiff base is reduced to the amine bond by mild reduction with sodium borohydride (NaBH ). Immediately after formation of the Schiff base in the above reaction, the reaction mixture is cooled to 4° in ice and 15 mg of NaBHi in 1.0 ml of KHCO3 at pH 7.9 is added. The reduction is allowed to occur in a cold-room at 4° for 12 hr. Since the reaction liberates hydrogen gas, reaction tubes are loosely capped to avoid a buildup of pressure. Unreacted [ S]MMPR-OP and NaBH are removed by dialyzing the reaction mixture against excess KHCO3 at pH. 7.9 until the radioactivity in the buffer drops to background levels. 

Chemical inhibition of L-phenylalanine ammonia lyase activity may be achieved by the use of typical carbonyl reagents such as sodium borohydride and potassium cyanide. Treatment of the enzyme with tritiated sodium borohydride and subsequent hydrolysis gave alanine in which the majority of the radioactivity was confined to the jj-methyl group . Similarly reaction with potassium cyanide and hydrolysis gave aspartic acid labelled exclusively in the -carboxyl group . These observations led to the proposal that the active site of the enzyme, like that of the related L-histidine ammonia lyase , contains a dehydro-alanine residue... 

Radioactive sodium-(3H)-borohydride and 45Ca++ were purchased from the Radiochemical Center, Amersham, England. 

Taxol analogs labeled at the 2-position have been prepared by Kingston and by Georg. The tritiated 2-(m-azidobenzoyl) analog 11.2.10 was prepared by reductive debromination of the corresponding 3-nitro-4-bromobenzoyl derivative with sodium [ H]-borohydride (393), and the corresponding non-radioactive analogs 11.2.11 and 11.2.12 were prepared by standard methods (388). 

Ribulosebisphosphate carboxylase (isolated by a published procedure ) (250 mg, 3.57 /imoles of protomeric unit > ) in 50 ml of metal-free 0.1 Af Bicine/60 inM potassium bicarbonate/0.1 mAf EDTA (pH 8.0) was treated at 25° with four successive 0.25-ml additions, at 20-min intervals, of 20 mAf Br-butanone- P2- Twenty minutes after the fourth addition, less than 10% of the initial activity (as determined by the method of Racker ) remained, and the reaction was terminated by the addition of 2-mercaptoethanol (10 mAf). A duplicate enzyme solution containing ribulosebisphosphate (1 mAf) was treated with reagent in an identical manner 95% of the initial enzymic activity was retained. A third enzyme solution under the same conditions but lacking both the reagent and substrate served as control. The three protein solutions were dialyzed against 0.1 M sodium chloride at 4° after dialysis the protein samples were made 0.1 M in sodium bicarbonate and 0.01 Af in sodium [ H]borohydride in order to reduce the carbonyl of the protein-bound reagent. The mixtures were maintianed in an ice bath for 30 min and then dialyzed exhaustively at 4° against 50 mAf sodium chloride. The samples then were assayed for protein concentration, radioactivity ( H and P), and sulfhydryl content (see the table). 

To determine the specific radioactivity of sodium [ H]borohydride, glutathione (30 /mmoles) and chloroacetol phosphate (20 /unoles) were incubated at room temperature for 30 min in 0.2 Af sodium bicarbonate. 

Chloroacetol phosphate alkylates the sulfhydryl group. ) The reaction mixture was cooled in an ice bath and then treated with a portion of the same stock solution of sodium [ Hjborohydride that was used in the reduction of the modified carboxylase (see above). The final borohydride concentration in the reaction mixture was 0.1 M. After 1 hr the mixture was acidified to pH 2.0 with 1 N HCl and chromatographed on a column (1.2 X 22 cm) of Dowex 50 (H ) equilibrated and eluted with 50 mAf HCl. The concentration of the glutathione derivative in the peak fraction was determined with the amino acid analyzer the radioactivity in the same fraction was also determined, thereby providing the specific radioactivity of the [ H] borohydride. 

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