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Covalent labelling of the Klenow fragment of DNA-polymerase i from E. coli

Degtiarev, S.K.; Zaĭchikov, E.F.; Lavruk, O.I.; Mitina, R.L.; Mustaev, A.A.; Rikhter, V.A.

Bioorganicheskaia Khimiia 15(10): 1356-1361

1989

ISSN/ISBN: 0132-3423
PMID: 2698620
Document Number: 343506
Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.

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