Fluorometric assay for avidin-biotin interaction.
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Anilino Naphthalenesulfonates↗
Biomedical subjects
P Horowitz
Publications and source records attributed to P Horowitz.
A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site.
The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.
Anilino Naphthalenesulfonates↗
The effects of lauryl maltoside on the reactivation of several enzymes after treatment with guanidinium chloride.
The present study confirms the previous reports that detergents can facilitate the reactivation of guanidinium chloride (GdmCl) denatured rhodanese (Tandon, S. and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618; Tandon, S. and Horowitz, P. (1987) J. Biol. Chem. 262, 4486-4491). Here, we report the effect of the detergent, lauryl maltoside, on the reactivation of several enzymes other than rhodanese. For this study we used five different enzymes each having a single polypeptide chain, namely: adenosine deaminase; 3-phosphoglyceric phosphokinase; myokinase; 3 alpha-hydroxysteroid dehydrogenase; and phosphoglucomutase. The regain of enzyme activity was used to monitor refolding. Like rhodanese, these enzymes were denatured in 6 M GdmCl and diluted into a buffer containing various concentrations of lauryl maltoside. The effect of lauryl maltoside on reactivating these proteins depended on the specific enzyme used. For example, in the presence of lauryl maltoside, reactivation of adenosine deaminase increased to 98%, while phosphoglucomutase could not be reactivated significantly. The critical micelle concentration (CMC) of lauryl maltoside was measured under the present experimental conditions using 2-(p-toluidinyl)naphthalene 6-sulfonate (TNS) as an apolar fluorescent probe, and gave a value of 0.085 mg.ml-1 in 10 mM sodium phosphate (pH 7.4). The reactivating effect of lauryl maltoside was not generally related to its CMC. In some cases an induction period was observed before the enzyme attained its steady-state velocity. This might suggest the presence of intermediate(s) in the refolding pathway that could have been stabilized by the detergent. These findings indicate that 'non-denaturing' detergents may be useful for assisting reactivation of enzymes, although the optimum conditions will have to be determined for each individual case.
Enzyme Inhibitors↗
Reversible thermal denaturation of immobilized rhodanese.
For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [35S]SO3(2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states.
Animals↗
Tritium exchange kinetics of yeast ribosomal subunits.
Tritium exchange kinetics of 60 S and 40 S ribosomal subunits from Saccharomyces cerevisiae were studied using a rapid centrifugal, ultrafiltration procedure. This assay used commercially available disposable columns and microconcentrators. The tritium-labeled ribosome was separated from the tritiated solvent using a prepacked gel-filtration column. The labeled ribosome was applied to a microconcentrator and the exchange-out kinetics of the ribosome was measured by centrifugation of the ribosome solution and measurement of the amount of radioactivity present in the filtrate. One major advantage of this method is its simplicity and rapidity. With this method, the tritium exchange-out behavior of 60 S and 40 S ribosomal subunits and of subunits during reassociation were determined. The two subunits exhibited different exchange-out rates. Both subunits consisted of multiple classes of exchangeable protons. Considerable conformational changes in both subunits were evident during subunit reassociation, as additions of equal molar quantities of unlabeled 40 S subunits to labeled 60 S subunits caused an immediate increase in the exchange rate. Similarly, an increase in the exchange rate in the small subunits upon addition of unlabeled 60 S subunits was observed.
Centrifugation, Density Gradient↗
Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effect of lauryl maltoside.
For the first time, the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) has been renatured from 6 M guanidinium chloride (GdmCl) by direct dilution of the denaturant at relatively high protein concentrations. This has been made possible by using the nonionic detergent dodecyl-beta-D-maltoside (lauryl maltoside). Lauryl maltoside concentration dependence of the renaturation and reactivation time courses were studied using 50 micrograms/ml rhodanese. There was no renaturation at lauryl maltoside (less than 0.1 mg/ml), and the renaturability increased, apparently cooperatively, up to 5 mg/ml detergent. This may reflect weak binding of lauryl maltoside to intermediate rhodanese conformers. The renaturability began to decrease above 5 mg/ml lauryl maltoside and was significantly reduced at 20 mg/ml. Individual progress curves of product formation, for rhodanese diluted into lauryl maltoside 90 min before assay, showed induction phases as long as 7 min before an apparently linear steady state. The induction phase increased with lauryl maltoside concentration and could even be observed in native controls above 1 mg/ml detergent. These results are consistent with suggestions that refolding of GdmCl-denatured rhodanese involves an intermediate with exposed hydrophobic surfaces that can partition into active and inactive species. Further, lauryl maltoside can stabilize those surfaces and prevent aggregation and other hydrophobic interaction-dependent events that reduce the yield of active protein. The rhodanese-lauryl maltoside complex could also form with native enzyme, thus explaining the induction phase with this species. Finally, it is suggested that renaturation of many proteins might be assisted by lauryl maltoside or other "nondenaturing" detergents.
Dose-Response Relationship, Drug↗
Low concentrations of guanidinium chloride expose apolar surfaces and cause differential perturbation in catalytic intermediates of rhodanese.
The conformations of sulfur-free and sulfur-containing rhodanese were followed with and without the detergent lauryl maltoside after guanidinium chloride (GdmCl) addition to 5 M to study the apparent irreversibility of denaturation. Without lauryl maltoside, sulfur-containing rhodanese denatured in a transition giving, at approximately 2.3 M GdmCl, 50% of the total denaturation induced change observed by activity, CD, or intrinsic fluorescence. Sulfur-free rhodanese gave more complex behavior by intrinsic fluorescence and CD. CD showed loss of secondary structure in a broad, complex, and apparently biphasic transition extending from 0.5 to 3 M GdmCl. The interpretation of the transition was complicated by time-dependent aggregation due to noncovalent interactions. Results with the apolar fluorescence probe 2-anilinonaphthalene-8-sulfonic acid, implicated apolar exposure in aggregation. Sulfhydryl reactivity indicated that low GdmCl concentrations induced intermediates affecting the active site conformation. Lauryl maltoside prevented aggregation with no effect on activity or any conformational parameter of native enzyme. Transitions induced by GdmCl were still observed and consistent with several phases. Even in lauryl maltoside, an increase in apolar exposure was detected by 2-anilinonaphthalene-8-sulfonic acid, and by protein adsorption to octyl-Sepharose well below the major unfolding transitions. These results are interpreted with a model in which apolar interdomain interactions are disrupted, thereby increasing active site accessibility, before the intradomain interactions.
Anilino Naphthalenesulfonates↗
The effects of the anilinonaphthalenesulfonates on the alkylation of tubulin: correlation between the appearance of sulfhydryl groups and apolar binding sites.
We have previously found that the sulfhydryl groups of tubulin are sensitive reporters of the effects of ligands on the tubulin molecule. In this study, we examined the effects of three anilinonaphthalenesulfonates on the interaction of tubulin with iodo[14C]acetamide and N, N'-ethylenebis(iodoacetamide). We found that 1,8-anilinonapthalensulfonate (1,8-ANS) and 2,6-anilinonaphthalenesulfonate (2,6-ANS) had no effect on the reaction with iodo[14C]acetamide. In contrast, bis(1,8-anilinonaphthalenesulfonate) (BisANS), an inhibitor of microtubule assembly, had a complex effect. Low concentrations of BisANS, where presumably only the high-affinity binding site was saturated, had little or no effect on alkylation. Higher concentrations of BisANS caused a strong enhancement of alkylation. None of these compounds had any effect on the reaction with N,N-ethylenebis(iodoacetamide). Our results suggest that the binding of BisANS, 2,6-ANS and 1,8-ANS to tubulin is complex and very different from that of the other anti-tubulin drugs. The correlation between the effects of drugs on alkylation of tubulin and the binding of BisANS is consistent with a model whereby the alkylatable sulfhydryls are located in apolar regions of the tubulin molecule.
Alkylation↗
Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect differential stabilization of the heme d1 and heme c moieties.
The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.
Chromatography, Gel↗
Characterization of rhodanese-tetracyanonickelate. An active site complex that slows sulfur-free rhodanese conversion to inert conformers.
The structure of the rhodanese-tetracyanonickelate (E X Ni(CN)2-4) complex has been characterized here in spectral and physical studies using urea as a structural perturbant. UV difference absorption, sedimentation velocity ultracentrifugation, fluorescence, and circular dichroism data show no significant conformational differences between sulfur-free rhodanese (E) and the E X Ni(CN)2-4 complex. The urea-induced enzyme structural transition curves were noncoincident when different structural parameters were monitored. For E, the urea concentrations giving half-maximal change (Cm) were: Cm = 3.0 M for activity measurement; Cm = 2.8 M for protein intrinsic fluorescence intensity; Cm = 4.3 M for ellipticity at 220 nm; and Cm = 3.3 M for wavelength of fluorescence emission maximum. For the E X Ni(CN)2-4 complex, Cm was shifted to a higher urea concentration relative to that found for E when activity (Cm = 3.6 M) and native protein fluorescence (Cm = 3.6 M) were the measured parameters but not when the wavelength of the emission maximum and ellipticity were monitored. Furthermore, urea-induced rhodanese structural changes were time-dependent and Ni(CN)2-4 binding on E slowed enzyme inactivation that is associated with structural relaxations. These findings, that Ni(CN)2-4 affects structural relaxations in rhodanese, are of particular interest in light of the recent suggestion that the E X Ni(CN)2-4 complex mimics a normally inaccessible intermediate in catalysis.
Binding Sites↗
A fluorometric assay for the biotin-avidin interaction based on displacement of the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid.
Avidin and biotin can be sensitively and accurately quantitated using the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS). In the presence of avidin, the fluorescence of 2,6-ANS is blue shifted with a large increase in quantum yield. Biotin binding causes complete displacement of the bound fluorophore with concomitant quenching of the fluorescence. The fluorometric monitoring of the displacement of 2,6-ANS can be used as a facile method of measuring the biotin-avidin interaction. 2,6-ANS displacement gives the same stoichiometry as the method using 4'-hydroxyazobenzene-2-carboxylic acid. Our initial studies of an affinity-purified avidin revealed that, of the four binding sites on the avidin tetramer, a mean of three remain available for biotin (or dye) binding; this finding highlights a caveat concerning the use of affinity-purified oligomeric-binding proteins with multiple sites. As compared with previous fluorescence methods, the use of 2,6-ANS gives high sensitivity without the necessity of preparing and purifying a covalent avidin conjugate. In addition, the present method;:is potentially more sensitive than those based on optical absorbance; uses a probe that has increased stability and a larger Stokes shift compared with fluorescein; is not subject to protein interference; and gives accurate results over a wide range of 2,6-ANS and avidin concentrations.
Anilino Naphthalenesulfonates↗
Studies on rhodanese synthesis in bovine adrenocortical cells.
The synthesis of adrenodoxin, a mitochondrial iron-sulfur protein required for adrenocortical steroidogenesis, is known to be regulated chronically by ACTH. Rhodanese, also a mitochondrial enzyme, is thought to be required for synthesis of iron-sulfur centers, such as those contained in adrenodoxin. In this study it has been found that rhodanese synthesis and activity are not regulated by ACTH, under the same conditions whereby ACTH induces adrenodoxin synthesis. In addition, unlike adrenodoxin, rhodanese is found to be synthesized in the mature form rather than as a higher molecular weight precursor protein.
Adrenal Cortex↗
Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions.
The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.
Ammonium Chloride↗
Bis(1,8-anilinonaphthalenesulfonate). A novel and potent inhibitor of microtubule assembly.
Two related compounds, 1,8-anilinonaphthalenesulfonate (1,8-ANS) and bis(1,8-anilinonaphthalenesulfonate) (Bis-ANS), are useful fluorescent probes for hydrophobic areas on protein molecules. Using fluorescence, we examined the binding of these compounds to bovine brain tubulin and found that Bis-ANS and 1,8-ANS bound to tubulin with Ki values of 2 and 25 microM, respectively. Bis-ANS potently inhibited the polymerization of tubulin into microtubules in vitro. In the presence of microtubule-associated protein 2, half-maximal inhibition of assembly was obtained at 3 microM Bis-ANS. In the presence of tau protein, half-maximal inhibition was obtained at 15 microM Bis-ANS. Surprisingly, 1,8-ANS, even at 200 microM, did not inhibit assembly. Scatchard analysis indicated one binding site for Bis-ANS on tubulin. Previous reports of 1,8-ANS binding to tubulin may have been influenced by the presence of Bis-ANS which until recently was a common contaminant of commercial supplies. Because of its intense fluorescence in addition to its potent inhibitory effects, Bis-ANS appears to be a useful probe to study microtubule assembly and other interactions involving tubulin.
Anilino Naphthalenesulfonates↗
The use of tritium exchange to detect conformational differences between intermediates in catalysis by the enzyme rhodanese.
Tritium-exchange experiments have been performed on enzyme forms related to obligatory catalytic intermediates in the rhodanese reaction: the free enzyme, E, and the sulfur-substituted enzyme, ES. Under the experimental conditions used, each form displayed tritium-exchange behavior that can be interpreted in terms of at least three classes of exchangeable sites. Class I with a t 1/2 approximately equal to 180 min and Class II with a t 1/2 approximately equal to 15 min were present in both E and ES. The conversion of ES to E has the effect of changing approx. 50 protons from the slow Class I behavior to exchange at a rate too fast to measure (Class III). These results are consistent with a conformational change on conversion of rhodanese from ES to E. The fact that a large number of protons are involved together with the very large change in exchange rate might indicate that extended areas of the protein change contact with the solvent, and would be compatible with a model for rhodanese catalysis that includes a coupled conformational change.
Animals↗
The use of intrinsic protein fluorescence to quantitate enzyme-bound persulfide and to measure equilibria between intermediates in rhodanese catalysis.
The intrinsic fluorescence of the enzyme rhodanese is quenched by as much as 30% when sulfur is transferred to the free enzyme form, E, giving the sulfur-substituted enzyme, ES. This fluorescence change (lambda ex = 295 nm and lambda em = 335 nm) has been used to quantitate the E and ES forms which are isolatable, obligatory intermediates in rhodanese catalysis. Fluorescence titration was performed using cyanide to irreversibly remove sulfur from ES. The results show a stoichiometry corresponding to 1 bound sulfur/molecule of the ES form of rhodanese (Mr = 33,000). The fluorescence changes were used to measure the concentrations of E and ES when these were in reversible equilibria induced by interactions with the substrates S2O3(2-) and SO3(2-). These results were compared with an equilibrium constant derived from published kinetic studies for the reaction (formula; see text) The very close agreement between the physical and kinetic methods indicate that there are no significant concentrations of intermediates other than E and ES. Overall, the results are compatible with the formation of a persulfide intermediate in rhodanese catalysis and are consistent with conclusions from x-ray crystallography and absorption spectroscopy. In addition, these procedures offer a facile method to measure equilibria between catalytic intermediates in the rhodanese reaction using functionally relevant concentrations.
Kinetics↗
Proteolytic interconversion of electrophoretic variants of the enzyme rhodanese.
It has been confirmed that the enzyme rhodanese, although a homogeneous single polypeptide chain protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is separable by electrophoresis under nondenaturing conditions into four species which differ in net surface charge (I-IV in the order of increasing positive charge). Limited proteolysis can interconvert these species. Chymotrypsin converts IV and III to II and forms a small amount of I. Carboxypeptidase B converts IV to III. The total protein among the species remains constant, and two-dimensional gels show that the change induced is below the resolution of the sodium dodecyl sulfate-polyacrylamide gel system. The suggestion that the products can be produced in the order IV, III, and II is supported by the results of sequential treatment of rhodanese first with carboxypeptidase B and then with chymotrypsin. It is concluded that there are covalent differences among the rhodanese species identified to date and an interconversion of forms can be triggered by proteolysis at the COOH-terminal end of the Mr = 33,000 single polypeptide chain which comprises the enzyme. This conclusion is strengthened by the close similarity between the amino acid composition of the peptide released by chymotrypsin and the composition expected on the basis of the known sequence. Furthermore, it appears that form IV is the primary in vivo product and the other species arise from it.
Animals↗
Constraints on the flexibility of bacteriorhodopsin's carboxyl-terminal tail at the purple membrane surface.
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Amino Acid Sequence↗