Biotoxin activity assays typically involve multistep sample preparation, multicomponent reactions, multistep analysis, or a combination thereof. We report a single-step, real-time ricin activity assay that requires little or no sample preparation and employs direct analysis in real time mass spectrometry. The release of adenine from the inhomogeneous substrate herring sperm DNA by ricin was determined to be 53 +/- 2 pmol adenine per picomole of ricin per hour. This procedure can be readily adapted to any enzyme for which a reactant or product of low molecular weight (up to approximately 600) can be identified.
Spin simulation and selective deuteration have been used to aid in the interpretation of 1D transferred nuclear Overhauser effect (TRNOE) NMR experiments on ricin B-chain/ligand systems. Application of these methods has revealed a change in the conformation of deuterated methyl beta-lactoside upon binding to the ricin B-chain which results in a slight change in glycosidic torsional angels which appear to dominate in the solution conformation. The combination of simulation and experiment also shows an important sensitivity of TRNOE magnitudes to dissociation rate constants and available spin-diffusion pathways for the ricin B-chain/ligand systems under study. The sensitivity to dissociation rates allows determination of rate constants for methyl beta-lactoside and methyl beta-galactoside of 50 and 300 s-1, respectively.
A lactate dehydrogenase (LDH) enzyme kinetics laboratory experiment has been developed in which students obtain kinetic data using a microplate spectrophotometer (reader). These instruments have the capability of reading absorbances of many samples in a very short time frame. In this experiment 12 samples are prepared at a time and the absorbances read in less than 1 min. In a 3-hr laboratory period, students collect data at five different substrate concentrations without inhibitor and also in the presence of two different concentrations of inhibitor. Students have enough time to repeat each part if they obtain too much scatter in their data. The enzyme examined, LDH, correlates with the study of metabolism and has particular relevance for students who are interested in medical careers. The LDH assay itself is not new, but the microplate format and the use of urea as a quench reagent are novel features. Students plot Michaelis-Menten and Lineweaver-Burk plots and calculate values for V(max) , apparent V(max) (V(max) (app) ), K(m) , apparent K(m) (K(m) (app) ), k(cat) , and K(I) . Students typically obtain results correctly showing that oxalic acid is a competitive inhibitor and oxamic acid is a noncompetitive inhibitor when lactate is the substrate of the reaction.
As predicted, a fibrillin domain forms a calcium-binding, EGF-like module. As the putative calcium-binding sites are found at the amino-terminal end of the modules, we propose that calcium ions may bind in the interfaces between domains, affecting the overall structure of the protein. The Arg to Pro mutation blocks domain folding in vitro, suggesting that lack of proper domain folding in vivo may contribute to the molecular defects responsible for Marfan syndrome.
Transferred nuclear Overhauser effect (TRNOE) experiments have revealed a change in the torsion angles about the alpha-1-6 glycosidic bond of methyl beta-melibioside upon binding of the melibioside to the ricin B-chain (Rb). A full relaxation rate matrix simulation of experimental buildup curves aided in quantitative interpretation of 1D selective inversion recovery TRNOE experiments. The data are consistent with a model in which both major (omega approximately 170 degrees) and minor (omega approximately -60 degrees) conformers for methyl beta-melibioside are significantly populated in solution while the Rb/methyl beta-melibioside complex has little of the minor conformer populated. The results indicate that the ricin B-chain excludes binding of certain ligand conformations on the basis of unfavorable interactions between the protein surface and remote portions of the disaccharide system.
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