Format

Send to

Choose Destination
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jul 15;1090:52-55. doi: 10.1016/j.jchromb.2018.05.022. Epub 2018 May 16.

Facile isolation of α-ribazole from vitamin B12 hydrolysates using boronate affinity chromatography.

Author information

1
Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
2
Department of Microbiology, University of Georgia, Athens, GA 30602, USA. Electronic address: jcescala@uga.edu.

Abstract

Alpha-ribazole (α-R) is a unique riboside found in the nucleotide loop of coenzyme B12 (CoB12). α-R is not an intermediate of the de novo biosynthetic pathway of coenzyme B12, but some bacteria of the phylum Firmicutes have evolved a two-protein system (transporter, kinase) that scavenges α-R from the environment and converts it to the pathway intermediate α-RP. Since α-R is not commercially available, one must either synthesize α-R, or isolate it from hydrolysates of vitamin B12 (cyano-B12, CNB12), so the function of the above-mentioned proteins can be studied. Here we report a facile protocol for the isolation of α-R from CNB12 hydrolysates. CNB12 dissolved in NaOH (5 M) was heated to 85 °C for 75 min, then cooled to 4 °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 °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 (α-R). Formic acid was removed by lyophilization, and the final yield of α-R was 85% from the theoretically recoverable amount. Methods for quantifying the concentration of α-R are reported.

KEYWORDS:

B(12) biosynthesis precursors; Boronate affinity chromatography; Late steps of coenzyme B(12) biosynthesis; α-Ribazole isolation

PMID:
29783174
PMCID:
PMC5977401
[Available on 2019-07-15]
DOI:
10.1016/j.jchromb.2018.05.022
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center