A dynamic dialysis method for studying protein-ligand binding using chromatographic theory

A new dynamic dialysis method has been developed for studying protein-ligand binding phenomena. The method depends on analysis of the elution pattern of ligand in a single dialyzing process where the ligand concentration in the sample compartment changes greatly with time. The dialyzer is composed of a long, narrow chamber (the sample compartment) between two sheets of semipermeable membrane and two outside chambers (the sink compartment) connected as a single path. Eluting buffer flows in the sink compartment to exchange the ligand with the solution in the sample compartment. Therefore, the ligand concentration gradient in the sink compartment is in the longitudinal direction. The mathematical expressions to analyze the experimental data were derived from a modified theory of chromatography. Examination of the binding of sulfanilamide to bovine serum albumin using this method shows that these equations are valid for use in studying protein-ligand binding.     

Microbore column exclusion chromatographic method for studying protein association and its relation with enzymatic activity

The principles of treating chromatographic data are considered to allow the determination of number of components, equilibrium and kinetic association constants of proteins by means of micro-column exclusion chromatography. The method is developed to study protein association in cases complicated by adsorption. The potentialities of the chromatographic method have been examined for the study of the mechanism of enzyme catalytic action.     

Kinetic analysis of the hydrodynamic transition accompanying protein folding using size exclusion chromatography. 2. Comparison of spectral and chromatographic kinetic analyses

The kinetics of the hydrodynamic volume change associated with the unfolding and refolding of a globular protein can be observed using high performance size exclusion chromatography. Chromatographic profiles that evidence such dynamics can be simulated using equations in which Chromatographic partitioning and the conformational transition are described in terms of a finite difference algorithm incorporating an apparent binding model to generate broad and asymmetric peaks. Application of these equations to the simple two-state unfolding transition of ribonuclease A in guanidine hydrochloride indicates that reliable kinetic parameters can be obtained using these equations. © 1993 John Wiley & Sons, Inc.     

A rapid method for determining kinetic parameters of enzymes exhibiting nonlinear thermal inactivation behavior

A rapid method is developed to analyze the kinetics of thermal inactivation of enzymes that exhibit a nonlinear biphasic log(activity)-time relationship. Thermal destruction experiments on alcohol dehydrogenase from baker's yeast demonstrate the applicability of the method. The method is based on physical considerations (as opposed to mathematical curve fitting/regression methods) and also serves as a quick check of results obtained using nonlinear regression. It is superior to fitting nonlinear enzyme inactivation data by first-order kinetics or taking the initial and final slopes of the inactivation data. In fact, the method is of general validity and can be applied to any decay process that can be represented by a sum of exponentials. (c) 1996 John Wiley & Sons, Inc.     

A method for studying insoluble immune complexes

Two variants of a method for determining the average composition of insoluble immune complex particles (IICP) are described. The first variant is based on measuring the specific turbidity (the turbidity per unit mass concentration of the dispersed substance) and the average size of IICP determined from dynamic light scattering (DLS). In the second variant, the slope of the logarithmic turbidity spectrum (wavelength exponent) is used instead of DLS particle size. Both variants allow the average biopolymer volume fraction to be determined in terms of the average refractive index of IICP. The method is exemplified by two experimental antigen+antibody systems: (i) lipopolysaccharide-protein complex (LPPC) of Azospirillum brasilense Sp245+rabbit anti-LPPC; and (ii) human IgG (hIgG)+sheep anti-hIgG. We have found that IICP can be modeled by incompact porous particles that contain about 30% of biopolymer substance and 70% of buffer.     

A kinetic proof-reading mechanism for protein sorting

Resident proteins of the exocytic pathway are maintained at various levels through coatomer protein I (COPI)-mediated recycling. Sorting of cargo by COPI requires GTP hydrolysis by ADP-ribosylation factor 1 (ARF-1). This small GTPase recruits coatomer onto Golgi membranes and upon hydrolysis, is thought to release coatomer back into the cytosol. This step requires the activating protein, ARFGAP1. By coupling sorting to a cargo-induced sequestering of ARFGAP1, we have formulated a kinetic proof-reading model that explains how a GTP hydrolysis-driven coat release can yield an active sorting event. The sorting scheme predicts a dependency on the amount of ARFGAP1 and explains the recent experimental findings that ARF-1 and COPI detach with different time constants from the Golgi membrane in vivo .     

A laboratory method for studying zooplankton swimming behaviors

A laboratory method is presented for studying zooplankton swimming behaviors such as phototaxis and photokinesis. The method attempts to standardize laboratory conditions and to minimize the effects of several phenomena which modify zooplankton behavior. The role of angular light distribution in zooplankton behavior is discussed, and an apparatus which simulates a natural underwater light environment is described. The procedure minimizes the fluctuations in zooplankton swimming speed and vertical distribution that are caused by large light stimuli, noise, food deprivation, endogenous rhythms, and other factors. The experimental animals were viewed remotely with the aid of a light amplifier and video camera. A mathematical equation and computer program for calculating three-dimensional swimming speeds of zooplankton from video recordings are described in detail.     

A reproducible method for studying three-dimensional knee kinematics

The methods used in movement analysis often rely on the definition of joint coordinate systems permitting three-dimensional (3D) kinematics. The first aim of this research project was to present a functional and postural method (FP method) to define a bone-embedded anatomical frame (BAF) on the femur and tibia, and, subsequently, a knee joint coordinate system. The repeatability of the proposed method was also assessed. Using FP method to define the BAFs, 4 kinematic parameters (flexion/extension, abduction/adduction, tibial internal/external rotation, and antero-posterior translation) were computed for 15 subjects walking on a treadmill. The repeatability for all four kinematic parameters was then assessed, using intra- and inter-observer settings. After pooling the results for all observers, the mean repeatability value ranged between 0.4 degrees and 0.8 degrees for rotation angles and between 0.8 and 2.2 mm for translation.     

A high-throughput method for enzyme kinetic studies

A simple and flexible setup for conducting drug metabolism studies is described in this report. A heating block was designed for the Multimek liquid handler platform for incubation of multiple samples at 37 degrees C in a 96-well format. This setup enables the rapid performance of drug metabolism experiments on a large number of samples. In this report, the authors present the validation of the system by 1) showing reproducible and consistent determination of the in vitro half-life of midazolam in every well across the entire plate and 2) determination of metabolic parameter values of midazolam, testosterone, diclofenac, warfarin, and dextromethorphan and inhibition parameter values of quinidine and ketoconazole, all comparable to literature values. In addition, the authors demonstrate the application of the setup to determining the metabolic stability of a set of proprietary compounds, the inhibition of activity of cytochrome P450 (CYP) enzymes, and the conduct of a single combination experiment that can simultaneously determine the metabolic stability and CYP inhibition activity. Overall, the system represents a simple, high-throughput and useful tool for drug metabolism screening in drug discovery.     

A tandem chromatographic column method for assaying cAMP-dependent protein kinase and protein kinase C with synthetic peptide substrates

A method was devised for assaying protein kinases that phosphorylate either Kemptide, such as cAMP-dependent protein kinase, or a glycogen synthase peptide, which is an excellent substrate for protein kinase C. Upon sequential processing of reaction mixtures through tandem columns of cation and anion exchange resins, radioactivity in background samples is nearly nil and the yield of phosphorylated peptides is high. This method reduces labor, radioactivity, enzyme requirements, and costs of assaying protein kinases.     

A microfluidic device with groove patterns for studying cellular behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 microm wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37 degrees C, 5% CO(2)). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.     

A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.     

A p.m.r. isotope-exchange method for studying the kinetic properties of dehydrogenases in intact cells.

A method to determine the activity of dehydrogenases in an intact-cell system is described. The method involves the use of n.m.r. to monitor bulk isotope exchange. The approach is illustrated by application to the isotope equilibration of pyruvate and lactate as catalyzed by lactate dehydrogenase in intact erythrocytes. Particular problems peculiar to bulk isotope exchange and its observation by n.m.r. are considered.