Effect of temperature on fluorescence and circular dichroism of Escherichia coli dihydrofolate reductase and its complexes.

Dihydrofolate reductase and its complexes have been studied by fluorescence and circular dichroism. NADPH, trimethoprim, pyrimethamine, or Methotrexate binding causes small changes in the enzyme far ultraviolet CD which possibly arise from alterations in polypeptide backbone of the enzyme; however, their effects on enzyme far ultraviolet CD are also explained as the result of ligand interactions with enzyme aromatic groups. In ternary complexes of the enzyme, fluorescence properties of bound NADPH are surprisingly sensitive to the type of inhibitor bound nearby. The effect of temperature on the enzyme and its complexes is clearly shown by changes in enzyme fluorescence and CD. At temperatures near 45 degrees C, the enzyme undergoes an irreversible denaturation, as shown by major alterations in enzyme far ultraviolet CD and by an increased rate of fluorescence quenching. Binary complexes with NADPH or Methotrexate stabilize the enzyme towards this heat denaturation, whereas bound trimethoprim and pyrimethamine do not. Ternary complexes with NADPH and any of the ligands are more stable than the enzyme itself toward heat denaturation. Fluorescence-temperature and fluorescence polarization studies show that near 30 degrees C the enzyme undergoes a reversible transition that is modified by NADPH or methotrexate.

Optical and hydrodynamic studies of the structure of bacteriophage f2. II. Fluorescence of the capsid.

Fluorescence properties of the icosahedral RNA virus bacteriophage f2 and its empty capsid are reported. Emission is dominated by tryptophan with a maximum wavelength of 320 nm for f2 and its empty capsid. In addition to this short wavelength maximum, perturbation and denaturation studies indicate the inaccessibility of the tryptophan residues. However a high degree of thermal quenching and a red shift in fluorescence emission on heating suggest a noncooperative structural transition, not a denaturation, which allows buried tryptophans to become exposed to solvent. Therefore the tryptophan residues may be located between subunits. Fluorescence from tyrosine is detected near 315 nm for both f2 and its empty capsid, and may indicate an unusual tyrosine environment. Sensitization of tryptophan fluorescence by tyrosine absorption and low values of polarization indicate tyrosine leads to tryptophan and tryptophan leads to tryptophan energy transfer. The presence of RNA in f2 decreases the efficiency of these transfer processes, but does not significantly affect the other reported fluorescence properties.

Comparison of colloidal gold electrode fabrication methods: the preparation of a horseradish peroxidase enzyme electrode.

In order to prepare biosensing electrodes which respond to hydrogen peroxide, horseradish peroxidase has been adsorbed to colloidal gold sols and electrodes prepared by deposition of these enzyme-gold sols onto glassy carbon using three methods: evaporation, electrodeposition and electrolyte deposition. In the latter method the enzyme-gold sol is applied to the surface of a glassy carbon disk electrode followed by an equal volume of 2 mM CaCl2. The electrolyte causes the sol to precipitate on the electrode surface, producing an immobilized enzyme electrode. Satisfactory electrodes which gave an electrochemical response to hydrogen peroxide in the presence of the electron transfer mediator ferrocenecarboxylic acid were produced by all three methods. Evaporation of horseradish peroxidase-gold sols produced electrodes with the best reproducibility and the widest linear amperometric response range. These electrodes can also easily be stored in a dry state. Although not as good as evaporation, electrodeposition also produced satisfactory electrodes. Electro-deposition provides the added advantage that it lends itself to the preparation of multi-enzyme/multi-analyte electrodes by the adsorption of different enzymes to separate gold sols, followed by sequential electrodeposition onto discrete areas of a multichannel electrode.

A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood.

The preparation of two immobilized enzyme electrodes is described. One electrode contains horseradish peroxidase absorbed to colloidal gold and deposited on a glassy carbon electrode along with cholesterol oxidase entrapped in a carrageenan hydrogel. The second electrode also includes cholesterol esterase entrapped in the carrageenan. The incorporation of ferrocene or ferrocenecarboxylic acid mediator is brought about by either evaporation on the glassy carbon electrode or, in the latter case, entrapment in the carrageenan hydrogel. Amperometric signal generation results from the HRP catalyzed turnover of H2O2, a secondary product of the cholesterol oxidase catalyzed oxidation of cholesterol. Use of these enzyme electrodes makes cholesterol detection possible in human serum, low density lipoprotein, and whole blood.

Mediator-free amperometric determination of toxic substances based on their inhibition of immobilized horseradish peroxidase.

We have demonstrated the feasibility of quantitatively detecting selected toxic materials through their inhibitory effect on an enzyme electrode that utilizes colloidal gold-immobilized horseradish peroxidase and does not require a mediator. Quantitative detection of azide, cyanide, thiourea, sulfide, and dichromate is demonstrated. The sensitivity and inhibition kinetics for this immobilized enzyme electrode are found to be different from those observed previously for homogeneous horseradish peroxidase. Possible reasons for this difference are discussed. Due to the availability of a large number of enzymes and their toxic inhibitors, this work based on immobilized enzyme inhibition coupled to an electrode surface significantly broadens the possible applications of biosensors and offers alternative methods for toxic substance determination.

Electrochemical enzyme immunoassay for detection of toxic substances.

Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We developed a new biosensor design that combines the advantages of immunoassay with electrochemical response. We established that this enzyme-linked immunosensor measures toxic substances in biological samples. The biosensor consists of two major elements: (1) an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents, and (2) the electronic components used in the signal readout. The result is an amperometric immunoassay based on coupling the immunochemical reaction to the enzyme electrode response by using a soluble, electrochemically active mediator. The specific question addressed was: Does the system's immunochemical detection reliably respond at sufficiently low analyte concentrations? We present our results in these areas: (1) enzyme immobilization on colloidal gold; (2) colloidal gold-enzyme deposition on the electrode surface; (3) mediator-antigen conjugate synthesis; (4) antibody incorporation at the electrode surface; (5) bioelectrode characterization and optimization; and (6) immunosensor demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as blood and urine and in environmental samples such as wastewater and drinking water.

Urea and guanidine hydrochloride induced dissociation and denaturation of bacteriophage F2 capsid.

A single, low molecular weight protein is found after urea or guanidine hydrochloride (Gdn.HCl) treatment of empty capsids derived from bacteriophage f2. The final product of denaturation is apparently a monomer, existing as a random coil in larger than or equal to 4.0 M Gdn.HCl but in a less extended form in 8.0 M urea. In contrast, an 11 S protein component is isolated after treatment of the intact virus with 4.0 M Gdn.HCl (Zelazo & Haschemeyer, 1969), indicating that RNA plays a role in stabilizing larger subunits. Denaturation by Gdn.HCl occurs in two stages as measured by changes in CD and Stokes radius: dissociation that involves a structural perturbation of aromatic side chains, followed by a major, cooperative transition that evidently results in the loss of all noncovalent structure. Denaturation by urea appears to be a much less cooperative process that occurs in several steps over a wide range of urea concentration (1--7 M). In both urea and Gdn.HCl, dissociation into subunits begins at a lower concentration of denaturant than the major changes in conformation.

Kinetic studies on the effect of the heme iron(III) on the protein folding of ferricytochrome c.

The three-dimensional conformation of ferricytochrome c results from specific folding of the polypeptide chain around the covalently bound heme so that His-18 and Met-80 are axially coordinated to the Fe(III). The Fe(III)-free, porphyrin protein has an intrinsic viscosity, sedimentation coefficient, and circular dichroism indicative of a compact, globular protein conformation comparable to the holoprotein. Both the porphyrin protein and ferricytochrome c are reversibly denatured by guanidinium chloride. Refolding of the porphyrin protein occurs in essentially a single, exceptionally rapid kinetic phase (tau = 14 ms, 0.75 M guanidinium chloride, pH 6.5, 25 degrees C); whereas refolding of ferricytochrome c occurs in two slower kinetic phases (TAU 1 = 0.10 S, TAU 2 = 20 S) UNDER COMPARABLE CONDITIONS. The presence of Fe(III) in the metalloporphyrin of ferricytochrome c thus has a major effect on the protein folding kinetics. The slow kinetic phase is evidently due to this effect of Fe(III) and not to the slow cis-trans isomerism of the peptide bond of proline residues as has been suggested.

Detection of intermediates in protein folding of carbonic anhydrase with fluorescence emission and polarization.

The three-dimensional structure of carbonic anhydrase is a result of specific folding of the protein chain to form a compact, globular molecule. Fluorescence measurements on the nature of the rate-limiting steps in folding from the random coil to the native structure show that each step involves an actual folding reaction of the protein chain. Emission intensity and polarization of the intrinsic fluorescence due to tryptophan residues reach a maximum during the early period of the folding process. The changes occur in at least three kinetic phases (tau1 less than 3 S, tau2 = 1 min, tau3 = 10 min, 1 M guanidinium chloride, 2 M NaC1, pH 7, 20 degrees C). None of these phases are explained by configurational changes in the fully unfolded chain. The results are consistent with a kinetic scheme that involves stepwise acquisition of the specific folded structure of the native enzyme.

Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I.

Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of fast reactions of carbon dioxide and bicarbonate in human erythrocytes measured by carbon-13 NMR.

The application of carbon-13 nuclear magnetic resonance spectroscopy to the study of the kinetics of millisecond timescale reactions of CO2 in human erythrocyte suspensions is described. The rates of intracellular enzyme catalyzed CO2 hydration and HCO3- dehydration were quantitatively determined, as well as the rates of CO2 diffusion into and out of the erythrocytes. The method also provides an accurate measure of the intracellular pH in the range of pH 6.0 to pH 7.0. A temperature dependence study was used to determine the thermodynamic functions for the intracellular hydration-dehydration reaction.

13-C nuclear magnetic resonance studies on the mechanism of action of carbonic anhydrase.

Binding of the substrate, bicarbonate, to bovine cobalt carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) has been studied with 13-C nuclear magnetic resonance. Two binding sites for bicarbonate have been identified. One loosely binds bicarbonate, inhibits p-nitrophenyl acetate activity, and must be the bicarbonate substrate binding site; the other tightly binds bicarbonate, is noninhibitory, and plays another role. Spinlattice relaxation times for the carbon atom of bicarbonate indicate that the substrate bicarbonate is bound directly to the metal center of the enzyme, while the other bicarbonate is bound in the outer coordination sphere of the metal. It is proposed that dehydration proceeds via HCO-3 minus coordinated directly to the metal center, while the outer sphere bicarbonate facilitates catalytically important proton transfers.

Kinetics of neutralization of bacteriophage f2 by rabbit gamma-G-antibodies.

The neutralization of bacteriophage f2 by intact gammaG-immunoglobulin or fragments is first order with respect to both bacteriophage and antibody. Minimum values for the rate constants are of the order of 10(7)M(-1) sec(-1). The temperature dependence of the rates corresponds to the activation parameters: DeltaHdouble dagger = 6.7 kcal mole(-1) and DeltaSdouble dagger = -4 cal deg(-1) mole(-1) (gammaG-immunoglobulin); DeltaHdouble dagger = 8.0 kcal mole(-1) and DeltaSdouble dagger = -0.9 cal deg(-1) mole(-1) (5S pepsin fragment); and DeltaHdouble dagger = 13.3 kcal mole(-1) and DeltaSdouble dagger = 12 cal deg(-1) mole(-1) (3.5S fragment). The kinetic observations are consistent with the view that the binding of a single antibody molecule can bring about phage neutralization. There are two ways in which a single antibody molecule can affect neutralization: (1) binding at or near some critical site on the phage may block its function, (2) binding may disturb the general architectural design of the protein shell of the phage. Although the rate of neutralization varied directly with antibody size, consideration of the activation parameters speaks against the dependence of neutralization on simple steric factors. Addition of antibodies directed against rabbit gammaG-immunoglobulin or the 5S pepsin fragment caused approximately a threefold neutralization enhancement. This enhancement may result from the detection of a class of infectious bacteriophage antibody complexes.

Mussel glue protein has an open conformation.

Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods.

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.

Proline isomerism in the protein folding of ribonuclease A.

The guanidinium chloride-unfolded state of ribonuclease A was found to be an equilibrium mixture of slow- and fast-refolding forms of the protein chain, as has been suggested. Both forms appear to have the same spectroscopic observables as judged by the relative changes in fluorescence emission and polarization. The equilibrium between them is thermally dependent, with deltaHapp equal to -1.4 kcal/mol. The activation energy Ea is equal to 18 kcal/mol. These findings are consistent with the proposal that cis-trans isomerism of peptide bonds that are NH2-terminal to proline residues is responsible for the slow phase of RNase A refolding. However, the actual dependence of the magnitude of the slow reaction on initial, prefolding temperature cannot be explained by a model in which the proline configurations of the fast refolding form must be identical to those of the native protein, as has been suggested. Instead, the data reveal that, although the native structure of RNase A contains two cis prolines, cis isomers need not be present in the fast-refolding form in order for folding to occur.

Kinetics and mechanism of refolding of bovine carbonic anhydrase. A probe study of the formation of the active site.

In kinetic studies of the folding of bovine carbonic anhydrase from disorganized to native structure, an azosulfonamide, 2-(4-sulfomylphenylazo)-7-acetamido-1-hydroxynaphthalene-3,6-disulfonate (I), has been used as a probe to follow the dynamics of formation of the active site region. The probe is a specific inhibitor of the native enzyme that binds in the active site crevice. The experiments, with previous data (Yazgan, A., and Henkens, R. W. (1972), Biochemistry 11, 1314), show that a tight binding site for I forms at an intermediate stage in the folding process. A subsequent conformational change perturbs the visible absorption and circular dichroism of bound I and could result in even tighter binding. The subsequent change completes formation of the active site. This is shown by results from separate experiments on the kinetics of recovery of activity (p-nitrophenyl acetate as substrate). Similar probe methods could be used with other proteins and enzymes to study the kinetics and mechanism of regeneration of specific sites--for example, the active site.

Kinetics and mechanism of dissociation of zinc ion from carbonic anhydrase.

The kinetics of dissociation of Zn2+ from the metalloenzyme carbonic anhydrase was measured over a range of pH, temperature, and acetate concentration. The rate of dissociation is extremely slow at neutral pH (t1/2 approximately 3) years, 4 degrees C), but increases in almost direct proportion to the hydrogen ion concentration and is enhanced in the presence of 1,10-phenanthroline or acetate. The thermodynamic stability of the zinc-apoenzyme complex was determined over a range of pH from rate data on binding and dissociation (stability constants 10(9)-10(11) M-1, 25 degrees C). The great stability of the complex and slow exchange of the apoenzyme ligand is attributed, at least in part, to the rigidity of the multidentate protein ligand.