Single-step bioluminescence analyses of enzymes, using Cibacrone Blue chromatography for removal of interfering dehydrogenases.
To provide for bioluminescence measurements of the enzymatic activities of dehydrogenases, disturbing contaminants were removed from a bacterial luciferase extract by chromatography, using Blue Sepharose CL-6B, a cross-linked agarose to which Cibacrone Blue F3G-A is covalently attached. This compound has a strong affinity to the dinucleotide fold, which is a region in enzymes binding NAD(H) or NADP(H). In contrast to the absorbed dehydrogenases, both luciferase and oxidoreductase were easily eluted and appeared close to the main bulk of UV-absorbing but analytically less important material. A rapid recording of the elution of luciferase was accomplished with a new electrochemical bioluminescence assay. Due to this and the early elution of the desired material, it could be chromatographed, recognized and collected in less than two hours. Thereby the light-yielding capacity of the sensitive material was well preserved. For bioluminescence assay solutions composed of pooled oxidoreductase-luciferase fractions, FMN and a long chain aldehyde were prepared and supplemented with NAD+ and either lactate, malate or 3-hydroxybutyrate. The analyses were carried out in a single step performance by adding the enzyme sample to the luciferase solution. Minute amounts of lactate dehydrogenase, malate dehydrogenase and 3-hydroxybutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the femtomole region. In case a dehydrogenase does not occur as a contaminant of a commercial luciferase preparation, purification with Cibacrone Blue can be omitted as demonstrated for glucose-6-phosphate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
D004136:Dinitrobenzenes; D004800:Enzymes, Immobilized; D006884:Hydroxybutyrate Dehydrogenase / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008156:Luciferases / Q000032:analysis; D008163:Luminescent Measurements; D008291:Malate Dehydrogenase / Q000032:analysis; D009243:NAD / Q000302:isolation & purification / Q000378:metabolism; D009249:NADP / Q000302:isolation & purification; D009578:Nitrobenzenes; D010088:Oxidoreductases / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives*
D004136:Dinitrobenzenes; D004800:Enzymes, Immobilized; D009578:Nitrobenzenes; C017842:cibacron blue-sepharose; D009243:NAD; D009249:NADP; C039275:N(4)-dinitrophenyl-2,4-diaminobutyric acid hydrazide; D012685:Sepharose; D010088:Oxidoreductases; D007770:L-Lactate Dehydrogenase; D006884:Hydroxybutyrate Dehydrogenase; D008291:Malate Dehydrogenase; D008156:Luciferases