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.

Application of Spectrophotometric Fingerprint in Cluster Analysis for Starch Origin Determination

The botanical origin of starch is of importance in industrial applications and food processing because it may influence the properties of the final product. Current microscopic methods are time-consuming. Starch consists of an origin-dependent amylose/amylopectin ratio. Triiodide ions bind characteristically to the amylose and amylopectin depending on the botanical origin of the starch. The absorbance of the starch-triiodide complex was measured for wheat, potato, corn, rye, barley, rice, tapioca and unknown origin starch, and within the different cultivars. Each starch sample had specific parameters: starch-triiodide complex peak wavelength maximum (λmax/nm), maximum absorbance change at λmax (ΔA) and λmax shift towards the unknown origin starch sample values. The visible absorption spectra (500-800 nm) for each starch sample were used as a unique fingerprint, and then elaborated by cluster analysis. The cluster analysis managed to distinguish data of two clusters, a cereal type cluster and a potato/tapioca/rice starch cluster. The cereal subclusters extensively distinguished wheat/barley/rye starches from corn starches. Data for cultivars were mostly in good agreement within the same subclaster. The proposed method that combines cluster analysis and visible absorbance data for starch-triiodide complex was able to distinguish starch of different botanical origins and cultivars within the same species. This method is simpler and more convenient than standard time-consuming methods.

Solvent Effects in Highly Efficient Light-Induced Molecular Aggregation

It has been reported that when irradiated with laser light non-resonant with the main absorption peaks, porphyrin molecules (4-[10,15,20-tris(4-sulfophenyl)-21,24-dihydroporphyrin-5-yl]benzenesulfonic acid, TPPS) in an aqueous solution become 10,000 to 100,000 times more efficient in light-induced molecular aggregation than expected from the ratio of gradient force potential to the thermal energy of molecules at room temperature. To determine the mechanism of this phenomenon, experiments on the light-induced aggregation of TPPS in alcohol solutions (methanol, ethanol, and butanol) were performed. In these alcohol solutions, the absorbance change was orders of magnitude smaller than in the aqueous solution. Furthermore, it was found that the absorbance change in the aqueous solution tended to be saturated with the increase of the irradiation intensity, but in the ethanol solution, the absorbance change increased linearly. These results can be qualitatively explained by the model in which intermolecular light-induced interactions between molecules within a close distance among randomly distributed molecules in the laser irradiation volume are highly relevant to the signal intensity. However, conventional dipole–dipole interactions, such as the Keesom interaction, are not quantitatively consistent with the results.

Homeopathic Potencies May Possess an Electric Field(-like) Component: Evidence from the Use of Encapsulated Solvatochromic Dyes

Background Homeopathic potencies have been shown to interact with a range of solvatochromic dyes to produce spectroscopic changes in the visible region of the electromagnetic spectrum. Furthermore, the nature of the changes observed under different experimental conditions is beginning to limit the number of possible hypotheses that can be put forward regarding the fundamental identity of potencies. Aims and Methods The present study uses β-cyclodextrins to encapsulate solvatochromic dyes of widely varying structures. The purpose of this approach is to de-couple the primary dye–potency interaction from any subsequent aggregation effects. Results Despite large differences in molecular structure between dyes, results show that potencies affect all dyes according to the same fundamental principles. Specifically, positively and negatively solvatochromic dyes collectively respond in opposite and complementary ways to potencies in accordance with the differential stabilisation of their excited and ground electronic states. Under the conditions of encapsulation, positively solvatochromic dyes display a bathochromic shift of, on average, 0.4 nm with a 2% absorbance change, and negatively solvatochromic dyes display a hypsochromic shift of, on average, 0.2 nm with a 1% absorbance change. This behaviour is only ever seen in two situations—where solvent becomes more polar or where an electric field is applied to solutions of dyes. Conclusions The conditions used in this and previous studies to investigate the interaction of potencies with solvatochromic dyes preclude increased polarity of solvent as being responsible for the observed effects and that an explanation in which potencies carry an electric field (or electric field-like) component is by far the more likely. From the magnitude of the spectral changes induced in the dye Brooker's merocyanine by Arsenicum 10M, an estimate of the strength of the postulated electric field of 1.16 × 107 V/m can be made, which is comparable with the potential difference across cell membranes.

Synthesis, Acid-Base Properties, Kinetics and Mechanism of Cu2+ Incorporation into Water-Soluble Highly Substituted Porphyrins

The synthesis, acid-base properties, and kinetics of Cu[Formula: see text] incorporation into water-soluble highly substituted porphyrins were studied. The basicity increased and the stepwise acid-base equilibrium was clarified by increasing the number of phenyl groups at the [Formula: see text] position, and the basicity of a dodeca-substituted porphyrin increased with the ionic strength. The metalation reaction of the dodeca-substituted porphyrin with Cu[Formula: see text] in aqueous solution revealed a biphasic absorbance change at 453 nm. Plots of [Formula: see text] or[Formula: see text] vs. the Cu[Formula: see text] concentration and of log ([Formula: see text] or[Formula: see text]/[Formula: see text] 0) vs. the ionic strength show that [Formula: see text] is dependent on the Cu[Formula: see text] concentration and ionic strength, while [Formula: see text] is independent of these parameters. These results confirm the stepwise metalation mechanism and the existence of an intermediate in aqueous solution, which is indicated by the biphasic absorbance change at 453 nm.

Study on the Antioxidation of Lac Dye

To investigate the in vitro antioxidation and antimicrobial activity of the lac dye, in this study, three free radicals DPPH·, ABTS+and O2ˉ· are used as the test objects to evaluate the free radial scavenging ability of the lac dye by comparing the absorbance change of free radical solution under specific wavelength. The results showed that, the lac dye has scavenging activity on DPPH· and ABTS+, but has no scavenging activity on O2ˉ·; when the lac dye reached a certain concentration, its scavenging rate on DPPH· and ABTS+can be as high as 68% and 87% respectively, with the antioxidant activity respectively 0.20 times and 3.42 times of that of the ascorbic acid, indicating that the lac dye is a kind of natural pigment of a certain scavenging activity on free radicals.

Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)–CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s−1. Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR–CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR–CYP3A4 complex can dissociate and reform between the first and second electron transfers.