alpha-chymotrypsin and 1-anilino-8-naphthalenesulfonate

In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the Förster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.

Topics: Anilino Naphthalenesulfonates; Animals; Binding Sites; Cattle; Chymotrypsin; Circular Dichroism; DNA; Ethidium; Humans; Male; Naphthalenesulfonates; Protein Binding; Salmon; Serum Albumin

We report studies of hydration dynamics at the surface of the enzyme protein bovine pancreatic alpha-chymotrypsin. The probe is the well known 1-anilinonaphthalene-8-sulfonate, which binds selectively in the native state of the protein, not the molten globule, as shown by x-ray crystallography. With femtosecond time resolution, we examined the hydration dynamics at two pHs, when the protein is physiologically in the inactive state (pH 3.6) or the active state (pH 6.7); the global structure and the binding site remain the same. The hydration correlation function, C(t), whose decay is governed by the rotational and translational motions of water molecules at the site, shows the behavior observed in this laboratory for other proteins, Subtilisin Carlsberg and Monellin, using the intrinsic amino acid tryptophan as a probe for surface hydration. However, the time scales and amplitudes vary drastically at the two pHs. For the inactive protein state, C(t) decays with an ultrafast component, close to bulk-type behavior, but 50% of the C(t) decays at a much slower rate, tau = 43 ps. In contrast, for the active state, the ultrafast component becomes dominant (90%) and the slow component changes to a faster decay, tau = 28 ps. These results indicate that in the active state water molecules in the hydration layer around the site have a high degree of mobility, whereas in the inactive state the water is more rigidly structured. For the substrate-enzyme complex, the function and dynamics at the probe site are correlated, and the relevance to the enzymatic action is clear.

Topics: Anilino Naphthalenesulfonates; Animals; Cattle; Chymotrypsin; Crystallography, X-Ray; Hydrogen-Ion Concentration; Kinetics; Ligands; Models, Molecular; Pancreas; Protein Conformation; Solutions; Water

An amino-terminal deletion mutant (residues 1-43) and a carboxy-terminal deletion mutant (residues 187-243) of human apoliprotein A-I (apo hA-I) have been produced from a bacterial expression system to explore the importance of the missing residues for the conformation of apo hA-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasticity of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo delta(1-43)A-I and apo delta(187-243)A-I have folded, tertiary structures. These structures differ in the specific arrangement of helical domains based, in part, on their relative thermodynamic stability, near- and far-UV CD, limited proteolysis, and the accessibility of tryptophans to fluorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo delta(187-243)A-I are very similar. Results from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical hairpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Apolipoprotein A-I; Chymotrypsin; Circular Dichroism; Fluorescent Dyes; Humans; Hydrolysis; Lipid Metabolism; Lipids; Protein Binding; Protein Conformation; Protein Folding; Recombinant Proteins; Sequence Deletion; Spectrometry, Fluorescence; Ultracentrifugation

Tubulin exhibits a number of characteristic functions that can be used to identify it. They include the ability to polymerize to microtubules, GTPase activity, and the binding of numerous antimitotic drugs and fluorophores. These functions can be differentially modified by low (0.1-1.0M) urea concentrations, and such urea-induced modifications are stable over time periods of minutes to hours. These intermediate states suggest the existence of restricted regions in the protein each of which is associated with a function and its own urea sensitivity. In order of decreasing sensitivity to urea these effects are decreased rate of polymerization of tubulin to microtubules > decreased extent of polymerization approximately decreased GTPase activity > enhanced fluorescence of a rapidly binding analogue of colchicine-MTPT [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] approximately decreased proteolysis by trypsin (after alpha Arg339) and by chymotrypsin (after beta Tyr281) > enhanced fluorescence of 1-anilino-8-naphthalenesulfonic acid (ANS). Additional evidence for the independent behavior of the restricted regions stems from the markedly different time dependence of the response to urea. These low urea concentrations do not induce significant changes in tryptophan fluorescence, suggesting that the observed effects are due to local unfolding. At higher urea concentrations (2-4 M), the enhanced fluorescence of the ligands is abolished; MTPT fluorescence decreases at lower urea concentrations than ANS fluorescence. Moreover, tubulin becomes highly susceptible to proteolysis at multiple sites, and tryptophan emission shows a red-shift, as expected. Multistep unfolding in response to denaturants has been reported for some other proteins. Tubulin appears to be an extreme example of such local responses that proceed under milder conditions than the global transition to the unfolded state.

Topics: Anilino Naphthalenesulfonates; Animals; Brain Chemistry; Chymotrypsin; Colchicine; Fluorescent Dyes; GTP Phosphohydrolases; Kinetics; Microtubules; Polymers; Protein Folding; Rats; Spectrometry, Fluorescence; Tropolone; Trypsin; Tryptophan; Tubulin; Urea

The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anilino Naphthalenesulfonates; Antibodies, Monoclonal; Binding Sites; Chymotrypsin; Fluorescent Dyes; Hydrolysis; Ligands; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Spectrometry, Fluorescence; Urokinase-Type Plasminogen Activator

A fluorescent study of some structural and functional properties of conjugates of a number of proteins (bovine serum albumin, pyruvate kinase, alpha-chymotrypsin, and the two toxic proteins of plant origin--ricin and viscumin) with polyalkylene oxides (polyethylene glycol and pluronic) has been carried out. Analysis of the intrinsic protein fluorescence showed that the structure and stability of various protein conjugates to denaturing agents change only slightly: the conformational mobility of tryptophan residues accessible to the solvent decreases, whereas that of tryptophan residues localized in the protein regions of low polarity remains unchanged. Besides, the conjugates display a higher thermal stability in comparison with their native proteins. The fluorescence of 1-anilinonaphthalene-8-sulfonic acid and water insoluble 2',3',4',5'-tetrabenzoylriboflavin bound to the native and modified proteins indicated that modification of the proteins with polyalkylene oxides decreased the polarity and increased the viscosity of the microenvironment. Hence, this modification makes it possible to change some functional characteristics of the protein without causing any significant changes in its structure.

Topics: Anilino Naphthalenesulfonates; Animals; Cattle; Chymotrypsin; Fluorescence; Fluorescent Dyes; Plant Preparations; Plant Proteins; Poloxalene; Polyethylene Glycols; Proteins; Pyruvate Kinase; Rabbits; Ribosome Inactivating Proteins, Type 2; Ricin; Serum Albumin, Bovine; Structure-Activity Relationship; Toxins, Biological

The preferential binding of 1-anilinonaphthalene-8-sulfonate by rabbit muscle phosphorylase a is the basis of a continuous fluorometric assay for phosphorylase kinase. The maximum rate of change in fluorescence (d delta F/dt) is dependent on both the concentration of phosphorylase kinase and on conditions, such as pH and calcium ion concentration, which affect the enzyme. Parallel measurements of the increases in fluorescence and of 32P incorporation demonstrate the existence of a distinct intermediate in the conversion of phosphorylase b to a. We have used the assay to monitor the increase in calcium-independent activity which accompanies the limited chymotryptic digestion of phosphorylase kinase.

Topics: Anilino Naphthalenesulfonates; Animals; Calcium; Chymotrypsin; Enzyme Activation; Fluorescence; Hydrolysis; Muscles; Phosphorylase a; Phosphorylase b; Phosphorylase Kinase; Rabbits