sepharose and 4-nitrophenyl-4--guanidinobenzoate

Guanidinobenzoatase, a plasma protein with possible application as a 'tumor marker', has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by 'controlled' immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 microlmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 micromol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, x220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.

Topics: Adsorption; Agmatine; Ascitic Fluid; Benzoates; Carboxylic Ester Hydrolases; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Humans; Indicators and Reagents; Ligands; Proteins; Sepharose

A new method, called affinity gel titration, for analyzing the specific interaction between an immobilized protein and a ligand molecule is presented. Only one or two experimental runs permit the determination of not only the equilibrium constant but also the amount of immobilized protein. A suspension of the immobilized protein on agarose gel beads is confined in a constant volume mixing cell. A solution of a specific ligand molecule of constant concentration is introduced into the cell so that its concentration in the cell increases continuously (as in a mixing chamber for forming a convex gradient). The correlation between the concentration of the ligand in the efflux and the cumulative volume of the efflux can be analyzed either by regression to a theoretical curve or by a graphical method. Specific binding of p-aminobenzamidine to immobilized Streptomyces griseus trypsin was studied by this method. The dissociation constant and the amount of active trypsin were determined. The values obtained were in good agreement with the inhibition constant obtained by a kinetic experiment with free trypsin and with the amount of active site measured by using p-nitrophenyl p'-guanidinobenzoate, respectively. A single run of the titration procedure could be completed within 1 h.

Topics: Autoanalysis; Benzamidines; Benzoates; Gels; Indicator Dilution Techniques; Kinetics; Ligands; Mathematics; Models, Chemical; Protein Binding; Proteins; Sepharose; Tosyllysine Chloromethyl Ketone; Trypsin

The first component of complement has been purified by using affinity chromatography on Sepharose-bound IgG. Unlike earlier procedures that yield the activated form of C1, in this method C1 is maintained in the native form by the protease inhibitor p-nitrophenyl, p'-guanidinobenzoate (NPGB). The procedure requires only two steps and yields pure C1 as judged both by SDS-PAGE analysis and by effective molecule calculations. The yields have varied from 30 to 50% in over 50 preparations. The functional properties of the purified native C1 correspond to those of C1 in serum. The dose-response activity profile is nonlinear, but becomes linear when C1 IS ALLOWED TO SELF-ACTIVATE. From SDS-PAGE analysis of the self-activated C1, all the C1r and C1s subcomponents are converted to the activated split products, indicating that all C1 molecules are biologically active. The recovery of C1 activity is dependent on the use of a heterologous source for the IgG on the affinity absorbant. The conditions of binding and elution from the Sepharose-IgG column are critical, indicating that immunoglobulin-bound C1 is rapidly inactivated under physiologic conditions by serum inactivators. The activation of the purified C1 in fluid phase has been explored both in the presence and absence of C1-inhibitor.

Topics: Benzoates; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Complement Activation; Complement C1; Complement C1 Inactivator Proteins; Electrophoresis, Polyacrylamide Gel; Guanidines; Hemolysis; Humans; Immunoglobulin G; Molecular Weight; Sepharose; Sucrose