Fluorescence analysis of hormone binding activities of wheat germ agglutinin

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.     

Fluorescence study of steroid hormone binding activity of Helix pomatia agglutinin

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin, found in the reproductive gland of a Roman snail. The present study has shown that HPA, in addition to its carbohydrate binding capacity possesses a hydrophobic binding activity. This protein binds with high affinity (k(D)=1.9-2.4 microM) steroid hormones: testosterone and progesterone, identified as putative ligands for the animal lectin HPA. Additionally, we have found that this lectin also interacts with adenine (k(D)=5.4+/-0.5 microM) and arylaminonaphthalene sulfonate TNS (k(D)=12+/-0.3 microM). Binding of HPA to hormones and adenine was accompanied by a significant increase of the intrinsic Trp fluorescence (up to 50%), characterizing the conformational changes in the lectin molecule. The hyperbolic shape of the binding curves indicated one high affinity site for the two steroid hormones and adenine, and more than one hydrophobic site for TNS, showed by the sigmoidal curve fit and Hill coefficient of (n(H)=1.5+/-0.2). Hormones and adenine compete for an identical binding site, suggested to occupy the central hydrophobic cavity of the HPA hexamer. Fluorescence resonance energy transfer (FRET) was applied to calculate the intramolecular distance between TNS and Trp chromophores.     

Human aldehyde dehydrogenase: coenzyme binding studies

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)     

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.     

Method dependence of apparent stoichiometry in the binding of salicylate ion to human serum albumin: a comparison between equilibrium dialysis and fluorescence titration.

The binding of salicylate ion to human serum albumin (HSA) was studied in 100 mM potassium phosphate buffer (pH 7.4, 25 degrees C), using equilibrium dialysis and fluorescence titration methods. The protein samples tested were (a) dialyzed human plasma and (b) a commercial preparation of HSA, essentially free of globulin and fatty acids. Independent of the analytical method used, Scatchard and nonlinear regression analyses of the data pointed to a single class of high-affinity salicylate binding sites. On the other hand, the binding parameters were found to be method dependent. K(d) ranged between 25 +/- 2.4 and 62 +/- 15 microM in equilibrium dialysis and between 10 +/- 1.3 and 40 +/- 3.0 microM in fluorescence titration. (The higher limits refer to plasma samples at high [HSA]). Following the same pattern, the apparent stoichiometry of binding (though independent of sample identity and concentration) was higher in equilibrium dialysis (n(app) = 3.2 +/- 0.10) than in fluorescence titration (n(app) 1.9 +/- 0.30). The difference between the two methods could be reconciled by invoking two distinct classes of binding sites (I and II), which had identical (or marginally different) K(d) values, while differing in the magnitude of the fluorescence signal (Deltaf) generated upon ligand binding (Deltaf, PL(I) = Deltaf(I); Deltaf, PL(II) = 0). Further, it was assumed that the state of occupation of class II sites affected the fluorescence efficiency of class I sites, such that Deltaf, PL(I,II) = betaDeltaf(I) (beta = interaction factor). A random binding scheme involving P, PL(I), PL(II), and PL(I,II) was formulated. The model adequately predicted the behavior of the system when monitored through the change in protein fluorescence: Taking K(d) = 25 microM and n(T) = 3, the interaction factor beta was found to be 0.62 +/- 0.10. It was concluded that the correct parameters for the binding of salicylate ion to HSA are K(d) = 25 +/- 2.4 microM and n(T) = 3.2 +/- 0.10, as indicated by equilibrium dialysis of purified HSA. Besides updating information relating to the salicylate binding potential of HSA, this study serves to illustrate a likely complication in the study of protein-ligand interactions by fluorometric methods.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

On the specificity of the D-galactose-binding lectin (PA-I) of Pseudomonas aeruginosa and its strong binding to hydrophobic derivatives of D-galactose and thiogalactose.

The D-galactose-binding lectin (PA-I) from the bacterium Pseudomonas aeruginosa, isolated by affinity chromatography on Sepharose, was examined for its relative affinities for simple sugars and their derivatives using equilibrium dialysis and hemagglutination inhibition tests. The lectin, which was found to bind 0.68 mol of D-galactose per subunit of 12.8 kDa, exhibited an association constant (Ka) of 3.4 x 10(4) M-1 for D-galactose and higher affinities for hydrophobic and thio derivatives of D-galactose (with highest affinity for the hydrophobic thio derivatives). alpha-Methyl-galactoside was a stronger inhibitor than the beta-methyl derivative and alpha-lactose was a weak inhibitor but the hydrophobic phenylated derivatives of the beta-configuration of D-galactose were more potent inhibitors than the respective alpha-galactosides.     

Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.

GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.     

Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.     

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Characterization and location of divalent cation binding sites in bovine glial fibrillary acidic protein

In our previous work [Yang, Z. W., & Babitch, J. A. (1988) Biochemistry (preceding paper in this issue)] divalent cations were found to be more effective promoters of astroglial filament formation than were monovalent cations. To determine if one or more divalent cation binding sites were the basis for this difference, glial fibrillary acidic protein (GFAP) was attached to nitrocellulose membranes and bathed in 1 microM 45CaCl2 in 60 mM KCl, 0.5 mM MgCl2, and 10 mM imidazole hydrochloride, pH 7.4. After removal of unbound 45Ca2+, GFAP was observed to bind calcium. Flow dialysis experiments showed that GFAP, dissolved in 2 mM Tris-HCl, pH 7.5, contained three classes of binding sites and 0.61 +/- 0.08 (SD), 1.7 +/- 0.4, and 4.6 +/- 0.2 sites per GFAP molecule with dissociation constants of 0.66 +/- 0.01 microM, 6.6 +/- 0.3 microM, and 44 +/- 1 microM, respectively. After addition of 0.5 mM MgSO4 to the flow dialysis solution, the high- and low-affinity sites were not observed while the remaining sites (1.95 +/- 0.15 per GFAP molecule) had a Kd = 2.16 +/- 0.25 microM. This showed that the high- and low-affinity sites are "Ca2+-Mg2+" sites while sites with intermediate affinity are calcium specific. To locate the calcium-binding regions, GFAP peptides were examined for calcium binding by calcium-45 autoradiography. The calcium-specific binding areas were localized in coil I. Computer-assisted analysis of the GFAP sequence revealed several EF-hand-like areas which could be the calcium binding sites. We conclude that divalent cations may play both structural and regulatory roles in astroglial intermediate filaments.     

Spectroscopic studies on Erythrina lysistemon seed lectin: effect of denaturing agents on lectin quaternary structure

The equilibrium unfolding of ElysL, a homodimeric legume lectin, was studied using different denaturing agents such as guanidinium chloride (GdnHCl), temperature and pH. Simultaneously, changes in the secondary as well as tertiary structure of lectin were followed by CD spectroscopy examination in both far and near-UV region, respectively. The hydrophobic cluster binding dye, 1-anilino-8-naphthalene sulfonate (ANS), was used to further explore intermediates and to follow the unfolding pathway of lectin. The adenine binding ability of lectin was examined and monitored via absorption spectra and the intrinsic tryptophan fluorescence. Our findings indicate that the ElysL unfolding process occurs via a three state pathway with an intermediate state. We also showed that ElysL binds adenine in a manner that involves a hydrophobic binding pocket that is independent of the carbohydrate binding sites.     

Heterologous overexpression of human NEFA and studies on the two EF-hand calcium-binding sites.

Human NEFA is an EF-hand, leucine zipper protein containing a signal sequence. To confirm the calcium binding capacity of NEFA, recombinant NEFA analogous to the mature protein and mutants with deletions in the EF-hand domain were expressed in Pichia pastoris and secreted into the culture medium at high yield. The calcium binding activity of each purified protein was measured by a modified equilibrium dialysis using the fluorescent Ca2+ indicator FURA-2 and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/mol NEFA was determined. The Ca2+ binding constants were resolved by intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca2+ binding sites with Kd values of 0.08 microM and 0.2 microM. Circular dichroism (CD) spectroscopy showed an increase from 30 to 43% in the amount of alpha-helix in NEFA after addition of calcium ions. Limited proteolytic digestion indicated a Ca2+ dependent conformational change accompanied by an altered accessibility to the enzyme.     

Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.     

Inhibition of firefly luciferase by alkane analogues

We reported that anesthetics increased the partial molal volume of firefly luciferase (FFL), while long-chain fatty acids (LCFA) decreased it. The present study measured the actions of dodecanol (neutral), dodecanoic acid (negatively charged), and dodecylamine (positively charged) hydrophobic molecules on FFL. The interaction modes are measured by (1) ATP-induced bioluminescence of FFL and (2) fluorescence of 2-(p-toluidino)naphthalene-6-sulfonate (TNS). TNS fluoresces brightly in hydrophobic media. It competes with the substrate luciferin on the FFL binding. From the Scatchard plot of TNS titration, the maximum binding number of TNS was 0.83, and its binding constant was 8.27 x 10(5) M(-1). Job's plot also showed that the binding number is 0.89. From the TNS titration of FFL, the binding constant was estimated to be 8.8 x 10(5) M(-1). Dodecanoic acid quenched the TNS fluorescence entirely. Dodecanol quenched about 25% of the fluorescence, whereas dodecylamine increased it. By comparing the fluorescence of TNS and bioluminescence of FFL, the binding modes and the inhibition mechanisms of these dodecane analogues are classified in three different modes: competitive (dodecanoic acid), noncompetitive (dodecylamine), and mixed (dodecanol).