Thiosulfate ion → Bromide ion

Participants

Reaction Steps

Hcy was further reacted with the BABA-activated BSA solutions prepared above. 20 mM 1-homocysteine was prepared by dissolving 1.5 mg of 1-homocysteine thiolactone in 0.5 ml 1M NaOH, incubating 10 minutes at 37° C. and finally diluting with 2.0 ml Na phosphate, pH 7.0, and 2.5 ml Na phosphate, pH 6.0 (final pH 7). Both BSA solutions prepared above were diluted to 4.0 ml with H2O and 1.0 ml of the Hcy solution was added to each and incubated overnight. The reactions were concentrated on a Centricon 30 and the reaction efficiency was estimated from the Br- concentration of the retained filtrates. Bromide standards were prepared by adding 2 μl of the appropriate KBr stock solutions to 98 μl of the -BABA-NHS reaction filtrate, producing standards with Br- concentrations of 0.0, 0.2, 0.4, 0.8, and 1.0 mM. 20 μl of the prepared Br- standards or 20 μl of the filtrate from the +BABA-NHS reaction were added to 0.9 ml of 0.1M Na citrate pH 5.5, followed by 20 μl of 0.8 mM fluorescein and 10 μl of 0.1M chloramine T. After 10 minutes, 20 μl of 25 mM thiosulfate in 0.2M Na2CO3 was added to stop the reaction and the absorbance recorded for each sample at 515 nm in a 1 cm cell. The absorbance values of the standards were plotted against the Br- concentrations to produce a standard curve, from which the concentration of Br- contained in the +BABA-NHS reaction was determined. From this value an estimate was obtained of the coupling efficiency of the BABA groups on BSA with Hcy. The +BABA-NHS reaction solution was concentrated 4 times on a Centricon 30, diluted with 1 ml 1×PBS (final conc. ~10 mg/ml) and set aside for subsequent antibody development.

Provenance & Evidence