Analytical Biochemistry Research Papers - Academia.edu (original) (raw)

Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free... more

Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processesin vivo.In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate

Ecto-5'-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5'-monophosphates yielding the... more

Ecto-5'-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5'-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [³H]adenosine-5'-monophosphate (AMP) as a substrate. The reaction product [³H]adenosine was separated from [³H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [³H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z' factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay.

Monomer detergent concentrations of Triton X-100, Chaps (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and sodium dodecyl sulfate in guanidine hydrochloride, formamide, and urea solutions were measured by an ultrafiltration... more

Monomer detergent concentrations of Triton X-100, Chaps (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and sodium dodecyl sulfate in guanidine hydrochloride, formamide, and urea solutions were measured by an ultrafiltration procedure. This simple and rapid procedure effectively separated the monomer forms of these detergents from their respective micelle forms. Critical micellar concentrations of these detergents in water measured by this procedure agreed well with previously reported values. Both ionic and nonionic chaotropic agents, e.g., guanidine hydrochloride, formamide, and urea, are demonstrated to significantly shift the equilibrium between the monomer and the micellar form of various detergents toward the direction of monomer in a concentration-dependent manner. Thus, monomer/micelle ratio of detergents in solution can be manipulated over a wide range by the concomitant use of chaotropic solvents. This has direct applications in experiments involving destruction of biomembranes and solubilization of hydrophobic molecules in aqueous solutions.

Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a... more

Understanding the intricate metabolic processes involved in plant cell wall biosynthesis is limited by difficulties in performing sensitive quantification of many involved compounds. Hydrophilic interaction liquid chromatography is a useful technique for the analysis of hydrophilic metabolites from complex biological extracts and forms the basis of this method to quantify plant cell wall precursors. A zwitterionic silica-based stationary phase has been used to separate hydrophilic nucleotide sugars involved in cell wall biosynthesis from milligram amounts of leaf tissue. A tandem mass spectrometry operating in selected reaction monitoring mode was used to quantify nucleotide sugars. This method was highly repeatable and quantified 12 nucleotide sugars at low femtomole quantities, with linear responses up to four orders of magnitude to several 100pmol. The method was also successfully applied to the analysis of purified leaf extracts from two model plant species with variations in th...

ABSTRACT A pH-rate method, based on following the time dependence of hydroxide release by monitoring the pH drift as a funetion of time, is developed and applied to the construction of a detailed log activity vs pH profile for yeast... more

ABSTRACT A pH-rate method, based on following the time dependence of hydroxide release by monitoring the pH drift as a funetion of time, is developed and applied to the construction of a detailed log activity vs pH profile for yeast pyruvate decarboxylase. In the pH range of 5 to 7 at 30°C this method reproduces the results of conventional initial rate studies but, in addition, produces a hitherto unreported pH independent activity region between pH 5.4-5.8. At least four pKs can be discerned in the pH-activity profile of this enzyme, instead of the two previously reported.

Most methods for analyzing real-time quantitative polymerase chain reaction (qPCR) data for single experiments estimate the hypothetical cycle 0 signal y0 by first estimating the quantification cycle (Cq) and amplification efficiency (E)... more

Most methods for analyzing real-time quantitative polymerase chain reaction (qPCR) data for single experiments estimate the hypothetical cycle 0 signal y0 by first estimating the quantification cycle (Cq) and amplification efficiency (E) from least-squares fits of fluorescence intensity data for cycles near the onset of the growth phase. The resulting y0 values are statistically equivalent to the corresponding Cq if and only if E is taken to be error free. But uncertainty in E usually dominates the total uncertainty in y0, making the latter much degraded in precision compared with Cq. Bias in E can be an even greater source of error in y0. So-called mechanistic models achieve higher precision in estimating y0 by tacitly assuming E=2 in the baseline region and so are subject to this bias error. When used in calibration, the mechanistic y0 is statistically comparable to Cq from the other methods. When a signal threshold yq is used to define Cq, best estimation precision is obtained by...

This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant... more

This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminum (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB mono-oxygenase reaction and the amount of protein used in the reaction. The kinetic behavior is characterized by K0.5=4.26x10(-...