RESUMO
Mass spectrometric analysis of reaction products allows simultaneous characterization of activities mediated by multifunctional enzymes. By use of MALDI-TOF mass spectrometry, the relative influence of magnesium and manganese promoted exonuclease and phosphatase activities of Esherichia coli exonuclease III have been quantitatively measured, offering a rapid and sensitive alternative to radioactivity quantification and gel electrophoresis procedures for determination of reaction rate constants. Manganese is found to promote higher levels of exonuclease activity, which could be a source of mutagenic effects if this ion were selected as the natural cofactor. Several potential applications of these methods to quantitative studies of DNA repair chemistry are also described.
Assuntos
Exodesoxirribonucleases/metabolismo , Metais/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Bases , Escherichia coli/enzimologia , Exodesoxirribonucleases/química , Exodesoxirribonucleases/efeitos dos fármacos , Exonucleases/efeitos dos fármacos , Exonucleases/metabolismo , Cinética , Magnésio/química , Magnésio/farmacologia , Manganês/química , Manganês/farmacologia , Metais/farmacologia , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Monoéster Fosfórico Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
Application of MALDI-TOF MS to direct sequencing of dsDNA substrates is demonstrated using a strategy that employs exonuclease III digestion of a target sequence. Experimental conditions for exonuclease III have been optimized for this application, including addition of essential divalent metal ion cofactors. A short cation-exchange column was designed to provide efficient sample cleanup and overcome major problems arising from salt interference.