Two new applications of the lately created technique of composition gradient static light scattering (CG-SLS) are presented. light scattering (CG-SLS), and demonstrated the ability of this solution to quickly identify and quantitatively characterize limited self-association equilibria in a remedy containing an individual protein component (3) and limited heteroassociation equilibria in a remedy containing two proteins components (4). The objective of this article would be to demonstrate two additional capabilities of this method: The simultaneous detection and quantitative characterization of both self- and hetero-association equilibria in a solution containing two protein components. The detection and characterization of indefinite self-association in a solution containing a single protein component. MATERIALS AND METHODS Materials Chymotrypsin and bovine pancreatic trypsin inhibitor (BPTI) were obtained from Sigma (St. Louis, MO), ENOX1 dialyzed against phosphate buffer, 0.05 M Na Phosphate + 0.2 M NaCl, previously titrated to the indicated pH value, and used without further purification. FtsZ, prepared in a buffer containing AZD8055 cost 50 mM Tris-HCl + 50 mM KCl + 0.1 mM GDP + 5 mM MgCl2, pH 7.5 (5), was a gift from Dr. Germn Rivas, CIB-CSIC. Protein concentrations were determined from the absorbance at 280 nm using the following standard values for absorbance in OD units/cm pathlength for a 1 g/l solution: chymotrypsin, 2.04 (6); bovine pancreatic trypsin inhibitor, 0.658 (7); and FtsZ, 0.345 (5). Refractive increments were determined as described in Attri and Minton (3), and found to be equal to 0.185 0.003 ml/g at 20C for all proteins utilized in this study. Immediately before light scattering measurement, solutions were prefiltered and centrifuged as described in Attri and Minton (3). Measurements of light scattering were carried out at 20C. Experimental procedures Experiments are conducted utilizing the apparatus described in Attri and Minton (4), and depicted schematically in Fig. 1. The apparatus consists of a programmable dual-syringe pump (Model No. 541C, Hamilton, Reno, NV) configured to deliver a solution of time-varying compositionthe composition gradientto the flow cells of a light scattering detector (DAWN-EOS, Wyatt Technology, Santa Barbara, CA) and an UV-visible absorbance detector (Model No. SM5100, Milton Roy, Miami, FL). The two flow cells are connected in parallel to provide simultaneous measurements of scattering and absorbance of volume elements of solution with identical composition throughout the composition gradient. Open in a separate window FIGURE 1 Schematic illustration of instrumentation used to perform composition gradient light scattering measurements: (and denotes the Rayleigh ratio, averaged from data obtained by multiple detectors, scaled to an optical constant defined in Attri and Minton (3), AZD8055 cost and as a function of 2, (6) where 1, such that , and obeying the conditions set out in the above definition of 1: (13) Thus, specification of the test values of the empirical parameters and together with = 100. It was verified that all series converged well before this limit.) RESULTS AND DISCUSSION Chymotrypsin (A) + bovine pancreatic trypsin inhibitor (B) Experiments were carried out over a range of pH values, in solutions prepared as described in Materials and Methods. The dependence of ?on fA obtained at three pH values is plotted in Fig. AZD8055 cost 2. Initially, unsuccessful attempts were made to analyze the composition gradient data in the context of a simple 1-1 hetero-association model, but it was soon noticed that derived ideals of the molar mass of chymotrypsin had been influenced by pH and unrealistically high at low pH ideals. Mention of the literature after that exposed that chymotrypsin may dimerize considerably under acid circumstances (8,11). (That is a graphic exemplory case of how the outcomes obtained utilizing the present technique can quickly information the investigator to the right selection of association model.) Subsequently, the info had been analyzed in the context of the model referred to by Eqs. 1C5 above. To get the maximum quantity of information regarding this two-component program, three distinct experiments.