New multi-step kinetics using common affinity biosensors saves time and sample at full access to kinetics and concentration

Today, affinity-based biosensorics is a standard technology in quantitative biomolecular interaction analysis, but suffers from low sample throughput and sometimes from inaccessible kinetics. A new methodology for such biosensors is introduced here that cuts down measurement time dramatically and increases confidentiality of results. In contrast to traditional applications, the ligand immobilized on the sensor chip is exposed to the binding analyte at a rapid stepwise change of the analyte concentration without the need for regenerations between analyte additions. In the application presented here, each addition of the analyte is succeeded by a buffer flow, yielding alternating association and dissociation phases in a "zigzag" style. This binding curve pattern is analyzed by means of novel fitting algorithms, which render detailed kinetics rate constants at a high level of self-consistency, and hence, validity due to multiple cross-checks. In comparison with traditional sequential kinetics analysis, this new multi-step kinetics approach returns practically identical (or improved) kinetics constants--at valuable savings in time/material since regeneration steps, ligand re-captures, or titration equilibrations are unnecessary.     

Analyzing a kinetic titration series using affinity biosensors

The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call "kinetic titration." This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.     

Interaction of human immunoglobulin G with l-histidine immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes

l-Histidine as pseudobiospecific ligand was immobilized onto poly(ethylene vinyl alcohol) hollow-fiber membranes to obtain an affinity support for immunoglobulin G (IgG) purification. The interaction of human IgG with the affinity membranes was studied by chromatography and equilibrium binding analysis. Adsorption was possible over a broad pH range and was found to depend strongly on the nature of the buffer ions rather than on ionic strength. With zwitterionic buffers like morpholinopropanesulfonic acid (Mops) and hydroxyethylpiperazineethanesulfonic acid (Hepes), much higher adsorption capacities were obtained than with other buffers like Tris-HCl and phosphate buffers. An inhibition analysis revealed that non-zwitterionic buffers competitively inhibit IgG binding, whereas Mops and Hepes in their zwitterionic form do not. By choosing the appropriate buffer system, it was possible to adsorb specifically different IgG subsets. The IgG molecules were found to adsorb on membrane immobilized histidine via their F_ab part. Determination of dissociation constants at different temperatures allowed calculation of thermodynamic adsorption parameters. Decrease in K_D with increasing temperature and a positive entropy value between 20 and 35°C (in Mops buffer) indicated that adsorption is partially governed by hydrophobic forces in that temperature range, whereas at lower temperatures, electrostatic forces are more important for adsorption.     

Sarkosyl is a good regeneration reagent for studies on vacuolar-type ATPase subunit interactions in Biacore experiments

Biacore is widely used for studies on protein–protein interaction in which regeneration is one of the most important steps. Here we introduce the anionic detergent sodium lauroyl sarcosinate (sarkosyl), which works satisfactorily as a regeneration reagent. After regeneration by the mild detergent, the subsequent binding experiment was reproducible without any degradation of the ligand. This regeneration condition can be employed for diverse combinations of ligand–analyte binding interactions and optimized as required.     

Baroresistant buffer mixtures for biochemical analyses

Hydrostatic pressure is a useful tool in the study of varied fields such as protein aggregation, association, folding, ligand binding, and allostery. Application of pressure can have a significant effect on the pK(a) values of buffers commonly used for biochemical analysis. Consequently, cationic buffers, rather than neutral ones, are generally used to minimize pH effects; however, even with these buffers, the change in pH over 3 kbar may be consequential in highly pH-sensitive biochemical systems. Using fluorescence-based assays, we have systematically examined the effects of pressure on various buffers in the neutral pH range. We show that many commonly used cationic and Good's buffers increase in pH with pressure on the order of 0.1 to 0.3 pH units/kbar, in agreement with other published values. Carboxylates and phosphate decrease in pH to a similar extent. Buffer mixtures, composed of both cationic and carboxylate or phosphate components, are shown to be an order of magnitude less pressure sensitive than the individual component buffers. Using various relative concentrations of Tris and either phosphate, tricarballylate (1,2,3-propanetricarboxylate), or CDA (1,1-cyclohexane diacetate) at pH values between 7 and 8 yields baroresistant buffer mixtures. Buffer mixtures can be optimized for a specific pH, and a list of mixtures is presented for general laboratory use.     

Bound and determined: a computer program for making buffers of defined ion concentrations.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.     

Effect of hydrogen ion concentration and buffer composition on ligand binding characteristics and polymerization of cow's milk folate binding protein

The ligand binding and aggregation behavior of cow's milk folate binding protein depends on hydrogen ion concentration and buffer composition. At pH 5.0, the protein polymerizes in Tris-HCl subsequent to ligand binding. No polymerization occurs in acetate, and binding is markedly weaker in acetate or citrate buffers as compared to Tris-HCl. Polymerization of ligand-bound protein was far more pronounced at pH 7.4 as compared to pH 5.0 regardless of buffer composition. Binding affinity increased with decreasing concentration of protein both at pH 7.4 and 5.0. At pH 5.0 this effect seemed to level off at a protein concentration of 10^–6 M which is 100–1000 fold higher than at pH 7.4. The data can be interpreted in terms of complex models for ligand binding systems polymerizing both in the absence or presence of ligand (pH 7.4) as well as only subsequent to ligand binding (pH 5.0).     

Inhibition of late endosome-lysosome fusion: studies on the mechanism by which isotonic-K+ buffers alter intracellular ligand movement.

Incubation of alveolar macrophages or hepatocytes in media in which Na+ is replaced by K+ ("isotonic-K buffer") inhibited the movement of internalized ligand from late endosomes to lysosomes (Ward et al.: Journal of Cell Biology 110:1013-1022, 1990). In this study we investigate the mechanism responsible for the isotonic-K+ block in movement of ligand from late endosomes to lysosomes. We observed that iso-K+ inhibition of endosome-lysosome fusion is not unique to alveolar macrophages or hepatocytes but can be seen in a variety of cell types including J774 and Hela cells. The inhibition in intracellular ligand movement was time dependent with the maximum change occurring after 60 minutes. Once established the inhibition resulted in a prolonged and apparently permanent decrease in vesicle movement. Cells were able to recover from the effects of iso-K+ buffers over a time course of 5-10 minutes when placed back in Na(+)-containing media. The effect of iso-K+ buffers was independent of intracellular pH changes and appeared to involve cell swelling. When cells were incubated in iso-K+ buffers under conditions in which cell volume changes were reduced, intracellular ligand movement approached normal levels. Such conditions included replacing Cl- with the less permeant anion gluconate, and by addition of sucrose to isotonic-K+ buffers. Analysis of the mechanism by which changes in cell volume could alter intracellular movement ruled out changes in cyclic nucleotides, Ca2+, or microtubules. These results suggest that changes in cell shape or volume can alter intracellular transport systems by novel routes.     

Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes

Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.     

Influence of herbaceous riparian buffers on physical habitat, water chemistry, and stream communities within channelized agricultural headwater streams

Herbaceous riparian buffers (CP 21 grass filter strips) are a widely used agricultural conservation practice in the United States for reducing nutrient, pesticide, and sediment loadings to agricultural streams. The ecological impacts of herbaceous riparian buffers on the channelized agricultural headwater streams that are common throughout the midwestern United States have not been evaluated. We sampled riparian habitat, geomorphology, instream habitat, water chemistry, fishes, and amphibians for 4 years from three channelized agricultural headwater streams without herbaceous riparian buffers and three channelized streams with herbaceous riparian buffers in central Ohio. Only seven of 55 response variables exhibited differences between buffer types. Riparian widths were greater in channelized headwater streams with herbaceous riparian buffers than streams without herbaceous riparian buffers. Percent insectivores and minnows were greater in channelized streams without herbaceous riparian buffers than streams with herbaceous riparian buffers. Percent clay, turbidity, specific conductance, and pH differed between buffer types only during one sampling period. No differences in geomorphology and amphibian communities occurred between buffer types. Our results suggest channelized agricultural headwater streams with and without herbaceous riparian buffers are similar physically, chemically, and biologically. Installation of herbaceous riparian buffers alone adjacent to channelized agricultural headwater streams in central Ohio and other parts of the midwestern United States may only provide limited environmental benefits for these stream ecosystems in the first 4-6 years after establishment. Alternative implementation designs combining the use of herbaceous riparian buffers with other practices capable of altering nutrient and pesticide loads, riparian hydrology, and instream habitat are needed.     

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.     

Calcium buffer injections delay cleavage in Xenopus laevis blastomeres

Microinjection of calcium buffers into the two-cell Xenopus laevis embryo delays cell division in a dose-dependent manner. Four calcium buffers in the BAPTA series with different affinities for calcium were used to distinguish between a localized calcium gradient regulating cleavage and the global calcium concentration regulating this event. DibromoBAPTA (Kd = 1.5 microM) was found to delay cleavage at the lowest intracellular concentration (1.3 mM) of the four buffers tested. The effectiveness of the calcium buffers was dependent upon the buffer dissociation constant but not in a linear fashion. The concentration of buffer required to delay cleavage increased as the buffer's dissociation constant shifted above or below that of the optimum buffer, dibromoBAPTA. This relationship between a calcium buffer's effectiveness at delaying cleavage and its calcium affinity provides support for the hypothesis that a calcium concentration gradient is required for normal cell cycle progression (Speksnijder, J. E., A. L. Miller, M. H. Weisenseel, T.-H. Chen, and L. F. Jaffe. 1989. Proc. Natl. Acad. Sci. USA. 86:6607-6611). DibromoBAPTA was also injected with two different amounts of coinjected calcium to test the possibility that the free calcium concentration of the buffer solution is the important parameter for delaying cleavage. However, we found that changes in buffer concentration have a much stronger effect than changes in the free calcium concentration. This observation supports the hypothesis that BAPTA-type buffers exert their effect by shuttling calcium from regions of high concentration to those of lower concentration, reducing any calcium concentration gradients present in the Xenopus embryo.     

Bicarbonate and other buffer systems can enhance the rate of H+ diffusion through mucus in vitro.

The effect of various diffusible buffers on mucus H+ permeability, and in particular the potency of the HCO3-/CO2 buffer system relative to other selected buffers is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layer was most pronounced for low DH+ values near neutrality. This concentration dependence was most marked with mucus layers in the buffer systems investigated. Furthermore, the nature and pKa values of the diffusible buffer systems used in this study had a profound effect on measured DH+. The effect was particularly striking in the case of HCO3- buffer with mucus. Possible implications of these in vitro findings in mucosal protection from acid are discussed.     

Buffer effects on EcoRV kinetics as measured by fluorescent staining and digital imaging of plasmid cleavage

We have developed a protocol to quantify polymer DNA cleavage which replaces the traditional radiolabeling and scintillation counting with fluorescent staining and digital imaging. This procedure offers high sensitivity, speed, and convenience, while avoiding waste and error associated with traditional 32P radiolabeling. This protocol was used to measure cleavage of pBR322 plasmid DNA by EcoRV, a type II restriction enzyme. EcoRV was found to exhibit an order of magnitude difference in binding in two apparently similar buffers used in previous investigations. To determine the origin of this effect, we measured reaction kinetics in buffers of different chemical nature and concentration: Tris, bis-Tris propane, Tes, Hepes, and cacodylate. We found that buffer concentration and identity had significant effects on EcoRV reaction velocity through large changes in specific binding and nonspecific binding (reflected in the Michaelis constant Km and the dissociation constant for nonspecific binding Kns). There were only small changes in Vmax. The source of the buffer effect is the protonated amines common to many pH buffers. These buffer cations likely act as counterions screening DNA phosphates, where both the protonated buffer structure and concentration affect enzyme binding strength. It appears that by choosing anionic buffers or zwitterionic buffers with a buried positive charge, buffer influence on the protein binding to DNA can be largely eliminated.     

Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase

While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the Mes [2-(4-morpholino)ethanesulfonic acid] buffer being used. In the presence of Mes, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H(2)O(2), was utilized. This anomaly seems to be due to the recycling of the phenolic substrate. The reaction is relatively inefficient, but at buffer concentrations of 10 mM or greater the recycling effect is nearly 100% with substrate concentrations less than 100 microM. The recycling effect is dependent on substrate structure, occurring with 4'-hydroxyacetophenone but not with 3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone). Characterization of the reaction parameters suggests that the phenoxyl radical from the peroxidase reaction interacts with Mes, causing the reduction and regeneration of the phenol. Similar responses occurred with related buffers such as Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and Pipes [piperazine-1,4-bis(2-ethanesulfonic acid)]. Results from this work and other reports in the literature indicate that great care is required in interpreting any results involving these buffers under oxidizing conditions.     

Buffering of calcium in the vicinity of a channel pore.

The function of calcium entry or release channels is often modulated by the cytosolic free calcium concentration. When such channels are studied in isolation, calcium buffer solutions are usually used to control the free calcium at the cytosolic face of the channel. Such solutions are generally formulated on the basis of equilibrium considerations. We calculate the gradient of [Ca2+] in the vicinity of a channel pore, in the presence of such buffers. We find that the effective degree of buffering near the pore is markedly affected by kinetic considerations. Commonly used EGTA solutions are completely ineffective in buffering [Ca2+] within macromolecular distances of the pore. In order to achieve useful buffering, the fastest buffers (e.g. BAPTA derivatives) must be used, in concentrations very much higher than those conventionally employed. Because of the diffusion limit on the maximum rate of binding of calcium to the buffer ligand, it is physically impossible to achieve good control of [Ca2+] at cytosolic levels at distances of less than a few nm from a pore conducting pico-ampere calcium current.     

Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

The binding of the opiate antagonist 3H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the "sodium effect" of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites.     

Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space

The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.     

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.     

Time courses of calcium and calcium-bound buffers following calcium influx in a model cell.

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.