Inorganic phosphate determination: Colorimetric assay based on the formation of a rhodamine B-phosphomolybdate complex

A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to P_i concentration in the precipitated complex, a molar absorption coefficient of 330,000 ± 5000 ⁻¹ cm⁻¹ (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol P_i with good precision, while quantities as low as 0.5 nmol P_i are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of P_i and total phosphate in (biological) samples.     

Influence of hydrophilic polymers on celecoxib complexation with hydroxypropyl beta-cyclodextrin

Complexation of celecoxib with hydroxypropyl beta-cyclodextrin (HPbetaCD) in the presence and absence of 3 hydrophilic polymers-polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG)-was investigated with an objective of evaluating the effect of hydrophilic polymers on the complexation and solubilizing efficiencies of HPbetaCD and on the dissolution rate of celecoxib from the HPbetaCD complexes. The phase solubility studies indicated the formation of celecoxib-HPbetaCD inclusion complexes at a 1:1M ratio in solution in both the presence and the absence of hydrophilic polymers. The complexes formed were quite stable. Addition of hydrophilic polymers markedly enhanced the complexation and solubilizing efficiencies of HPbetaCD. Solid inclusion complexes of celecoxib-HPbetaCD were prepared in 1:1 and 1:2 ratios by the kneading method, with and without the addition of hydrophilic polymers. The solubility and dissolution rate of celecoxib were significantly improved by complexation with HPbetaCD. The celecoxib-HPbetaCD (1:2) inclusion complex yielded a 36.57-fold increase in the dissolution rate of celecoxib. The addition of hydrophilic polymers also markedly enhanced the dissolution rate of celecoxib from HPbetaCD complexes: a 72.60-, 61.25-, and 39.15-fold increase was observed with PVP, HPMC, and PEG, respectively. Differential scanning calorimetry and X-ray diffractometry indicated stronger drug amorphization and entrapment in HPbetaCD because of the combined action of HPbetaCD and the hydrophilic polymers.     

Colorimetric determination of orthophosphate in the assay of inorganic pyrophosphatase activity

Using Baginski and Zak's (12) method for determining inorganic orthophosphate as a starting point, a number of conditions which influence the accuracy and precision of the determination of pyrophosphatase activity have been shown: nonenzyme-catalyzed acid hydrolysis by protein precipitation agents and molybdate-catalyzed hydrolysis of pyrophosphate together with interference in the determination of orthophosphate by the substrate pyrophosphate and other components from the reaction mixture, Tris, chloride, acetate, citrate, boric acid. With regard to these sources of error, a method is described for determining pyrophosphatase activity, and its reliability is investigated.     

Colorimetric assay for lignin peroxidase activity determination using methylene blue as substrate

Summary A colorimetric method for determination of lignin peroxidase activity has been developed which is based on the demethylation of methylene blue. The use of this dye shows the practical advantage of using a wavelength in the visible region of spectrum. The method can be efficiently used for the enzyme quantification as its sensitivity is close to the veratryl alcohol assay.     

Fluorometric studies on inclusion complexation of L/D-tryptophan by beta-cyclodextrin 6-O-pyridinecarboxylates

Novel beta-cyclodextrin (beta-CD) derivatives, bearing a nicotinic or isonicotinic moiety, have been synthesized by a convenient method in 21 and 25% yields, respectively. The stability constants (K) and Gibbs free energy changes (-DeltaG degrees ) for the inclusion complexation of beta-cyclodextrin 6-O-mono(3-pyridinecarboxylate) (1), 6-O-mono(4-pyridinecarboxylate) (2), and 6-O-monobenzoate (3) with L- and D-tryptophan have been determined by spectrofluorome try in aqueous buffer solution (pH = 7.20) at 25.0 degrees C. All of the modifications dramatically enhanced the original K for beta-CD by a factor of 30-280 and interestingly switched the original enantiomer preference for L- to D-tryptophan, thus affording the inverted enantio-selectivities of K(L)/K(D) = 2.5 for beta-CD and K(D)/K(L) = 1.2-2.1 for the modified CDs 1-3. These results are discussed from the viewpoints of the size-fit and geometrical complementary relationship between the host and guest.     

Enhanced bioavailability of soy isoflavones by complexation with beta-cyclodextrin in rats

In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 microg x min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 microg x min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD).     

Investigation of different modifications to the tetrazolium based colorimetric viability assay

Summary Three commonly used solvents for the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based viability assay for mammalian cells were compared: Acid/propan-2-ol, dimethyl sulfoxide (DMSO) and a lysing buffer containing sodium dodecylsulphate (SDS). Acid/propan-2-ol and DMSO could only be used when most of the medium was removed from the cell suspensions, whereas the lysing buffer was found to perform satisfactorily for both hybridomas and fibroblast cell lines without medium removal for cell densities up to 10⁶ cells mL⁻¹. Furthermore a newly synthesized tetrazolium salt was investigated, which forms a water soluble formazan upon reduction and thus eliminates the need for a solvent. However this salt adds a new complication to the method: the need for an electron carrier. For this reason we do not find that the new tetrazolium salt has any practical advantages over MTT.     

Colorimetric assay method for determination of the tannin acyl hydrolase (EC 3.1.1.20) activity

A new colorimetric method of tannase (tannin acyl hydrolase, EC 3.1.1.20) assay has been developed using its specific substrate tannic acid. It is based on the changes in optical density of substrate tannic acid after enzymatic reaction at 530 nm. The residual tannic acid was measured by a modified BSA precipitation method. This assay is very simple, reproducible, and very convenient, and with it tannase activity can be measured in relation to the growth of the organism.     

比色法测定硫酸小诺霉素注射液含量

硫酸小诺霉素注射液的含量测定 ,目前卫生部药品标准采用生物检定法[1] ,该法受培养基、菌种、温度、时间等影响较大 ,也有报道用高效液相气谱法 ,但因此法检验仪器昂贵 ,普及率不高 ,推广受到限制。根据氨基糖甙类抗生素与铜离子在碱性条件下形成稳定的蓝色络合物[2 ] ,在 5 6     

Colorimetric enzymatic activity assay based on noncrosslinking aggregation of gold nanoparticles induced by adsorption of substrate peptides

The mechanisms of colorimetric assays based on aggregation of gold nanoparticles (GNPs) have been separated into two categories, crosslinking, and noncrosslinking aggregation. The noncrosslinking aggregation has recently been emerging as a simple and rapid mechanism and has been applied to enzymatic activity assays and DNA detection. We report here the detailed study of an enzymatic activity assay for protein kinases based on noncrosslinking aggregation. The principle of the assay is to detect kinase activity by utilizing the difference of coagulating ability of a cationic substrate peptide and its phosphorylated form toward GNPs with anionic surface charge. The critical coagulation concentrations (CCCs) of the peptides were about 10(3) times lower than those of the metal cations with the same cationic charges. The multivalent coordination bonds of the functional groups of the peptides with the GNP surface will strongly support the adsorption of the peptide on the GNP surface. The effect of the GNP size (10, 20, 40, 60 nm) on the dynamic range of OD before and after aggregation was studied. The dynamic range became a maximum for 20 nm GNP among those studied. The difference of CCC between the phosphorylated and nonphosphorylated peptides was governed by (1) the ratio between the peptide concentration and the surface area concentration of GNP and (2) the net charge of the peptides. When the assay system was applied to the activity assessment of protein kinase A, the dynamic range of OD was largest for 20 nm GNPs. However, when the peptide concentration was lowered, the largest 60 nm GNP was advantageous because of its smaller specific surface area.