Optimization of a Colorimetric Assay to Determine Lactate Dehydrogenase B Activity Using Design of Experiments

Lactate dehydrogenase B (LDH-B) is overexpressed in lung and breast cancer, and it has been considered as a potential target to treat these types of cancer. Herein, we propose a straightforward incomplete factorial (IF) design composed of 12 combinations of two reaction buffers, three pH values, three salt (NaCl) concentrations, and three incubation times, which we called IF-BPST (Buffer/pH/Salt/Time), for the optimization of a colorimetric LDH-B assay in a final volume of 100 µL using 96-well plates. The assay is based on the absorbance change at ~570 nm and the color change of the reaction mixture due to the release of NADH that reacts with nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS), resulting in the formation of a blue-purple formazan. The results obtained using the IF-BPST were comparable with those obtained by response surface methodology. Our work revealed that the NBT/PMS assay with some modifications can be used to measure the activity of LDH-B and other dehydrogenases in a high-throughput screening format at the early stages of drug discovery. LDH-B containing lysates cannot be assayed directly, however, due to the sensitivity of the method toward detergents. Thus, we suggest precipitating the proteins in the lysates to remove the interfering detergents, and then to dissolve the protein pellet in a suitable buffer and carry out the assay.

In vitro examination of oral tissue conditioners potential toxicity

The oral tissue conditioners are applied temporarily to the gingival surface of a denture for the purpose of reconditioning the abused denture supporting tissues, allowing them to return to a normal, healthy state. According to chemical composition they can be classified into methacrylic and silicon conditioners. The objective of this research was to examine the potential toxicity of one, seven and thirty day extracts of different concentrations. Different effective concentrations (5, 12.5, 25 and 50%) of two silicone and four methacrylic conditioners extracts were used after extraction period of one, seven and thirty days. Amount of toxic substances and dynamics of their release were monitored by high-pressure liquid chromatography (HPLC). HPLC analysis could not be applied to the silicone conditioners because they consist of polymeric and inorganic 19 substances. Evaluation of cell proliferation, as cytotoxic parameters, was done by Mosmann?s MTT assay based on reduction of yellow tetrazole (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - MTT) to purple formazan by mitochondrial succinate dehydrogenase of metabolic active cells. With increasing of extraction period, the amount of released potential toxic substances increased. The cytotoxicity of tested materials significantly increased with extract concentration increase and duration of extraction period. To improve the biological characteristics of mobile dentures relined by soft materials, soaking in water the day before insertion into patient?s mouth was recommended. Silicone conditioners showed less cytotoxicity compared to the methacrylic conditioners, so they are more appropriate for daily dental practice.

A Colorimetric Assay to Quantify Dehydrogenase Activity in Crude Cell Lysates

Nitroblue tetrazolium (NBT) in the presence of phenazine methosulfate (PMS) reacts with the NADPH produced by dehydrogenases to produce an insoluble blue-purple formazan. Endpoint assays taking advantage of this reaction have been successfully used to detect the activity of several dehydrogenases. Here we present a version of this assay suitable for determining the kinetics of 6-phosphogluconate dehydrogenase catalysis in crude lysates of bacterial cells prepared in 96-well plates. Using the assay to screen a small library of variant 6-phosphogluconate dehydrogenases generated by error-prone polymerase chain reaction, we were able to identify three variants with improved activity and thermostability over the parent enzyme. These enzymes were partially purified and shown to be expressed at higher levels than the parent (leading to the increase in activity), and all three variants were indeed more thermostable than the parent (temperature midpoints 4-7°C higher) after purification. Thus the NBT-PMS assay appears suitable for screening libraries of variant dehydrogenases.

A Rapid Electrophoretic Method for the Determination of the Isoenzymes of Serum Lactate Dehydrogenase

A convenient electrophoretic procedure for the separation and quantitation of lactate dehydrogenase (LDH) isoenzymes is described. The system uses polyacetate Sepraphore III strips.* The areas of activity are shown by incubation with an LDH substrate combined with tetra-nitro-blue-tetrazolium. The reduction of the latter to the purple formazan is quantitatively related to the enzyme activity. Quantitative determination of the individual colored areas is performed by densitometry.