Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 1090, Issue -, Pages 52-55Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2018.05.022
Keywords
alpha-Ribazole isolation; B-12 biosynthesis precursors; Boronate affinity chromatography; Late steps of coenzyme B-12 biosynthesis
Funding
- National Institutes of Health [R37 GM040313]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R37GM040313, R01GM040313] Funding Source: NIH RePORTER
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Alpha-ribazole (alpha-R) is a unique riboside found in the nucleotide loop of coenzyme B-12 (CoB12). alpha-R is not an intermediate of the de novo biosynthetic pathway of coenzyme B-12, but some bacteria of the phylum Firmicutes have evolved a two-protein system (transporter, kinase) that scavenges alpha-R from the environment and converts it to the pathway intermediate alpha-RP. Since alpha-R is not commercially available, one must either synthesize alpha-R, or isolate it from hydrolysates of vitamin B-12 (cyano-B-12, CNB12), so the function of the above-mentioned proteins can be studied. Here we report a facile protocol for the isolation of alpha-R from CNB12 hydrolysates. CNB12 dissolved in NaOH (5 M) was heated to 85 degrees C for 75 min, then cooled to 4 degrees C for 30 min. The solution was neutralized with HCl (5 M), and the hydrolysate was diluted with an equal volume of ammonium acetate (0.3 M, pH 8.8). Alkaline phosphatase was added and the mixture was incubated at 37 degrees C for 16 h. After incubation, the sample was loaded onto a boronate affinity resin column, washed with ammonium sulfate (0.3 M, pH 8.8), water (to remove residual corrinoids) and finally with formic acid (0.1 M) to release (alpha-R). Formic acid was removed by lyophilization, and the final yield of alpha-R was 85% from the theoretically recoverable amount. Methods for quantifying the concentration of alpha-R are reported.
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