sepharose and 1-anilino-8-naphthalenesulfonate

Guanylyl cyclase-activating proteins (GCAPs) and recoverin are retina-specific Ca(2+)-binding proteins involved in phototransduction. We provide here evidence that in spite of structural similarities GCAPs and recoverin differently change their overall hydrophobic properties in response to Ca(2+). Using native bovine GCAP1, GCAP2 and recoverin we show that: i) the Ca(2+)-dependent binding of recoverin to Phenyl-Sepharose is distinct from such interactions of GCAPs; ii) fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) is markedly higher at high [Ca(2+)](free) (10 microM) than at low [Ca(2+)](free) (10 nM) in the presence of recoverin, while an opposing effect is observed in the presence of GCAPs; iii) fluorescence resonance energy transfer from tryptophane residues to ANS is more efficient at high [Ca(2+)](free) in recoverin and at low [Ca(2+)](free) in GCAP2. Such different changes of hydrophobicity evoked by Ca(2+) appear to be the precondition for possible mechanisms by which GCAPs and recoverin control the activities of their target enzymes.

Topics: Anilino Naphthalenesulfonates; Animals; Calcium; Calcium-Binding Proteins; Cattle; Eye Proteins; Fluorescence Resonance Energy Transfer; Guanylate Cyclase-Activating Proteins; Hippocalcin; Hydrophobic and Hydrophilic Interactions; Lipoproteins; Nerve Tissue Proteins; Protein Binding; Protein Conformation; Protein Isoforms; Recoverin; Retina; Sepharose; Spectrometry, Fluorescence; Tryptophan

Urease was chosen as a model multimeric protein to investigate the utility of reversible denaturation for immobilization to a hydrophobic support. Of the various procedures investigated, acidic denaturation provided the highest degree of immobilization and enzymatic activity with lowering of Km (apparent). Exposure of hydrophobic clusters in the protein molecule induced by the acidic pH environment was confirmed by fluorescence studies using 8-anilino-1-naphtalene-sulfonate as a hydrophobic-reporter probe. The catalytic potential of the enzyme at low pH values was dramatically improved with significant heat and pH stability enhancement on immobilization. Furthermore, the immobilized preparation was used successfully in continuous catalytic transformations. Based on the results presented in this article and a recent report involving a relatively more simple monomeric protein, it is suggested that reversible denaturation may be of general utility for immobilization of proteins, which are not normally adsorbed on hydrophobic supports.

Topics: Anilino Naphthalenesulfonates; Catalysis; Enzyme Stability; Enzymes, Immobilized; Fabaceae; Fluorescent Dyes; Hydrogen-Ion Concentration; Protein Denaturation; Sepharose; Temperature; Urease

125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied. 125I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19 +/- 0.53 x 10(-6) M and 2.69 +/- 0.78 x 10(-5) M, respectively. The number of binding sites of the high and the low affinity sites were 1.28 +/- 0.46 mol/mol and 23.5 +/- 9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of 125I-T4 to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T4 binding to HSA-Sepharose.

Topics: Anilino Naphthalenesulfonates; Bilirubin; Binding Sites; Binding, Competitive; Fatty Acids; Humans; Iodine Radioisotopes; Protein Binding; Sepharose; Serum Albumin; Thyroxine

Binding of the microtubule protein, tubulin, to hydrophobic groups immobilized on octyl sepharose has been investigated. The results indicate that tubulin binds to octyl sepharose in a time-, temperature-, and concentration-dependent manner. Binding is multiphasic, with one fast phase and at least two slow phases, and is influenced by the presence of antimitotic drugs. Colchicine, vinblastine and podophyllotoxin enhance the fast binding of tubulin with very little effect on the slow binding. Pre-incubation of tubulin with the apolar probe, bis(1,8-anilinonaphthalenesulfonate) (BisANS) enhances both the rapid and slow phases of binding of tubulin to octyl sepharose. 1,8-Anilinonaphthalenesulfonate, the monomer of BisANS, has no effect. These results are consistent with a model for tubulin decay which involves the appearance of hydrophobic sites with time.

Topics: Anilino Naphthalenesulfonates; Colchicine; Podophyllotoxin; Sepharose; Tubulin; Vinblastine

The present study revealed that the IgG immunoglobulins of normal or non-immune rabbit IgG and anti-bovine serum albumin, anti-ovalbumin, anti-bovine IgG and anti-p-chlorobenzoic acid antibodies could non-specifically bind to bis-(p-chlorophenyl)-acetic acid (DDA) coupled to omega-amino-hexyl Sepharose (AHS) by the use of strengthened hydrophobic interactions dependent on the concentration of NaCl. The main binding site on the adsorbent of DDA-substituted AHS (DDA-AHS) was found to be the DDA ligand. The hydrophobic potency of the DDA ligand was thought to be more effective than that of p-chlorobenzoic acid coupled to AHS. Out study also demonstrated that two different binding sites capable of interacting the DDA ligand were contained in IgG molecules. One was located in the Fc region and the other in the Fab region. The former had an ability to adhere to the DDA-AHS adsorbent in the presence of NaCl of 3 M or over, while the latter showed heterogeneous binding behavior depending upon its antibody specificity. Differences in the chromatographic distribution among the whole IgG immunoglobulins including anti-DDA antibody were found by a hydrophobic salting-out chromatography (HSOC) method on a DDA-AHS column. It was therefore assumed that when whole IgG proteins were subjected to HSOC on a DDA-AHS column, the hydrophobic binding site in the Fc region played a decisive role at high salt concentrations of 3 M or over, while the hydrophobic binding site in the Fab region played a major role at intermediate and low salt concentrations of 2 M or below. Thus, by taking advantage of this HSOC method, whole IgG or its Fab molecules possessing very strongly hydrophobic binding sites to promote high quantum yields of 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence can be easily separated. We concluded that the ligand of DDA is a probe for the hydrophobic regions in IgG immunoglobulins.

Topics: Anilino Naphthalenesulfonates; Animals; Binding Sites, Antibody; Chromatography, Agarose; Chromatography, Gel; DDT; Fluorescent Dyes; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin Fragments; Immunoglobulin G; Ligands; Papain; Rabbits; Sepharose