LC-EC determination of nucleotides and nucleic acids: application to enzyme assays and the analysis of DNA fragments.
High-performance liquid chromatography (HPLC) coupled to an electrochemical detector in an oxidative mode was used to analyze purine bases, nucleosides, and nucleotides as well as restriction fragments of nucleic acids. Ligands were separated by liquid chromatography with electrochemical detection (LC-EC) using size exclusion, ion-exchange, or reverse phase techniques. Using an amperometric electrochemical detector the determination was characterized with respect to sensitivity, selectivity, and capacity factor. It was observed from hydrodynamic and cyclic voltammetry that the optimum oxidation potential differed for the three major classes of purines, permitting an enhancement in selectivity when compared to detection. Guanylyl moieties demonstrated a half-wave potential at 0.800 V vs Ag/AgCl, while those for the adenylyl and inosylyl groups are above 1,000 V vs Ag/AgCl. The facility of the method to analyze components of a complex biological milieu was demonstrated by examining the purine pools of crude and partially purified eye lens homogenates as well as by comparing the traditional hexokinase assay to the newly developed LC-EC technique. Additionally, LC-EC was compared to detection for determination of the purine-metabolizing enzyme activities, adenylate deaminase and adenylosuccinate synthetase from crude cellular lysates of the cellular slime mold, Dictyostelium discoideum. Finally, the technique was used to assay the fragments from lambda-DNA cut with the restriction endonuclease Pst-1.
Malliaros
D P
DP
Department of Chemistry, University of Lowell, Massachusetts 01854.
DeBenedetto
M J
MJ
Guy
P M
PM
Tougas
T P
TP
Jahngen
E G
EG
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D004563:Electrochemistry; D004798:Enzymes / Q000032:analysis*; D009696:Nucleic Acids / Q000032:analysis*; D009711:Nucleotides / Q000032:analysis*; D010084:Oxidation-Reduction; D011687:Purines / Q000032:analysis / Q000378:metabolism
D004798:Enzymes; D009696:Nucleic Acids; D009711:Nucleotides; D011687:Purines; D004247:DNA; D004262:DNA Restriction Enzymes; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific