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  • Title: [Covalent labelling of the Klenow fragment of DNA-polymerase I from E. coli].
    Author: Degtiarev SKh, Zaĭchikov EF, Lavruk OI, Mitina RL, Mustaev AA, Rikhter VA.
    Journal: Bioorg Khim; 1989 Oct; 15(10):1356-61. PubMed ID: 2698620.
    Abstract:
    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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