Thermal Inactivation of Glucose Oxidase

Thermal inactivation of glucose oxidase (GOD; β-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 °C. The activation energy increased from 60.3 kcal mol–1 to 72.9, 76.1, and 88.3 kcal mol–1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal·mol–1·deg–1 in the presence of 7.1 × 10–5m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25–90 °C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 °C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 °C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure. Thermal inactivation of glucose oxidase (GOD; β-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 °C. The activation energy increased from 60.3 kcal mol–1 to 72.9, 76.1, and 88.3 kcal mol–1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal·mol–1·deg–1 in the presence of 7.1 × 10–5m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25–90 °C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 °C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 °C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure. Glucose oxidase (β-d-glucose:oxygen-oxidoreductase, EC 1.1.3.4) from Aspergillus niger is a flavoprotein that catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor. Glucose oxidase (GOD) 1The abbreviations used are: GOD, glucose oxidase; ANS, 8-anilino-1-naphthalenesulfonic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); HPLC, high performance liquid chromatography. finds application in the food and fermentation industry apart from being an analytical tool in biosensors for medical applications and environmental monitoring (1Röhr M. Kubicek C.P. Kominek J. Rehm H.J. Reed G. Biotechnology. 3. Verlag Chemie Weinheim, Muenchen1983: 455-456Google Scholar, 2Turner A.P.F. Karube I. Wilson G.S. Biosensors: Fundamentals and Applications. Oxford University Press, Oxford1987: 770Google Scholar, 3Gouda M.D. Thakur M.S. Karanth N.G. Biotechnol. Lett. 1997; 11: 653-655Google Scholar). This protein is a dimer of two identical subunits with a molecular weight of 160,000 (4Tsuge H. Natsuakai O. Ohashi K. J. Biochem. 1975; 78: 835-843Google Scholar). The dimer contains two disulfide bonds, two free sulfhydryl groups (4Tsuge H. Natsuakai O. Ohashi K. J. Biochem. 1975; 78: 835-843Google Scholar), and two FAD molecules (tightly bound) not covalently linked to the enzyme (5Swoboda B.E.P. Biochim. Biophys. Acta. 1965; 175: 365-369Google Scholar). The dimer has a high degree of localization of negative charges on the enzyme surface and dimer interface (6Ahmed A. Akhtar M.S. Bhakuni V. Biochemistry. 2001; 40: 1945-1955Google Scholar). The flavin cofactors are responsible for the oxidation-reduction properties of the enzyme (7Jones M.N. Manley P. Wilkinson A. Biochem. J. 1982; 203: 285-291Google Scholar). Under denaturing conditions, the subunits of GOD dissociate accompanied by the loss of cofactor FAD (7Jones M.N. Manley P. Wilkinson A. Biochem. J. 1982; 203: 285-291Google Scholar, 8O'Malley J.J. Weaver J.L. Biochemistry. 1972; 11: 3527-3532Google Scholar). Various additives such as salts, mono- and polyhydric alcohols, and polyelectrolytes were used to increase the thermal stability of GOD (9Ye W.N. Combes D. Manson P. Enzyme Microb. Technol. 1988; 10: 498-502Google Scholar, 10Appleton B. Gibson T.D. Woodward J.R. Sens. Actutor B. 1997; 43: 65-69Google Scholar). The effectiveness of additives depended on the nature of enzyme, its hydrophobic/hydrophilic character, and the degree of its interaction with the additives (9Ye W.N. Combes D. Manson P. Enzyme Microb. Technol. 1988; 10: 498-502Google Scholar). Aggregation, the main causative factor for the inactivation of glucose oxidase, could be prevented by modifying the microenvironment of the enzyme (11Ye W.N. Combes D. Biochim. Biophys. Acta. 1988; 999: 86-93Google Scholar). The thermal stability of GOD at 60 °C could be increased by incorporating lysozyme as an additive during immobilization. The role played by the complementarity of surface charges of the enzyme and lysozyme appeared to be crucial in the stabilization of GOD (12Gouda M.D. Thakur M.S. Karanth N.G. Electroanalysis. 2001; 13: 849-855Google Scholar). The presence of salt ions (primarily sulfate) is known to increase the stability of the folded conformations of proteins (13Baldwin R.L. Biophys. J. 7. 1996; 1: 2056-2063Google Scholar). Details of the mechanism are not yet completely understood, partly because of the presence of several intra- and intermolecular interactions in proteins that may or may not be stabilized by sulfate. Light is yet to be shed on the mechanism of thermal inactivation of GOD, despite several attempts at improving its stability (9Ye W.N. Combes D. Manson P. Enzyme Microb. Technol. 1988; 10: 498-502Google Scholar, 10Appleton B. Gibson T.D. Woodward J.R. Sens. Actutor B. 1997; 43: 65-69Google Scholar, 11Ye W.N. Combes D. Biochim. Biophys. Acta. 1988; 999: 86-93Google Scholar). An understanding of the thermal inactivation mechanism of GOD could lead to thermostabilization of the enzyme using appropriate additives. With this objective, experiments were carried out on the effect of some selected additives on the thermal stability of GOD. In addition to lysozyme, found earlier by us to increase the stability of GOD, two more salts, NaCl and K2SO4, which are commonly known to stabilize enzymes through ionic and hydrophobic interactions, respectively, were selected for the thermal stability studies reported here. The mechanism of inactivation and the effect of additives on the thermal stability of the enzyme were followed by kinetics of inactivation, spectroscopic measurements, and size exclusion chromatography. GOD (EC 1.1.3.4) from A. niger (type VII-S, 180,000 units/g solid), FAD, acrylamide, and N,N′-methylene-bisacrylamide, SDS, and lysozyme from hen's egg and 8-anilino-1-naphthalenesulfonic acid (ANS) from Sigma Chemical Co., β-mercaptoethanol, glycine, TEMED, and horseradish peroxidase (320 PPG units/mg) from ICN Biomedical Inc., Ohio, USA, and Shodex® PROTEIN KW-803 size exclusion column (300 × 8 mm) with an exclusion limit of 1.5 × 105 from Showa Denko, Japan were used. All other chemicals and buffer salts used were of analytical grade. Purification of GOD—The traces of catalase, associated with commercial preparations of GOD, were removed by size exclusion chromatography on a Sephacryl S-200 HR column (45 × 2.1 cm) preequilibrated with 20 mm phosphate buffer (pH 6.0). GOD was loaded on the column, and 0.5-ml fractions were collected at a flow rate of 10 ml/h. Protein concentration and activity of the fractions were measured. The protein concentration (of GOD) was determined using a value of A280 nm1%=13.8(14Swoboda B.E.P. Massey V. J. Biol. Chem. 1965; 240: 2209-2215Google Scholar). The fractions containing GOD were pooled and used. Enzyme Activity Assay—GOD was assayed at 30 °C by peroxidasecoupled assay (15Bergmeyer H.U. Methods Enzymat. Anal. 1974; 3: 457-458Google Scholar). Glucose and peroxidase were added to an o-dianisidine containing buffer (pH 6.0). GOD solution, appropriately diluted, was added after proper mixing. The increase in absorption at 460 nm was monitored for 4 min at 30 °C with a spectrophotometer. SDS-PAGE—SDS-PAGE experiments were performed on 17.5% vertical minislab gel (Broviga, Balaji Scientific Instruments, Chennai, India) according to Laemmli (16Laemmli U.K. Nature. 1970; 277: 680-685Google Scholar). Gels were fixed using water:methanol:trichloroacetic acid (5:4:1 by volume) and stained with 0.1% w/v Coomassie Brilliant Blue in water:methanol:acetic acid in the same ratio. Gels were destained in water:methanol:acetic acid in the above mentioned ratio until the background was clear. Molecular weight protein markers (Bangalore Genei, India) used were phosphorylase b (97,400), bovine serum albumin (66,000), ovalbumin (43,000), carbonic anhydrase (29,000), soybean trypsin inhibitor (20,000), and lysozyme (14,300). Thermal Unfolding Transitions by Activity Measurements—The loss of enzyme activity as a function of temperature was followed, in the presence and absence of additives, in 20 mm phosphate buffer (pH 6.0). The enzyme samples were incubated for 15 min at different temperatures ranging from 25 to 80 °C. After cooling to 4 °C, the residual activity was measured at 30 °C by transferring an aliquot to the assay mixture. The midpoint of thermal inactivation, Tm, at which the activity was diminished by 50%, was calculated from the plot of percent residual activity versus temperature. Kinetics of Thermal Inactivation—Kinetics of thermal inactivation of GOD was studied at different temperatures (56 – 67 °C), both in the absence and presence of selected additives. 100 μl (1 mg/ml) of enzyme solution was added to 0.9 ml of 20 mm phosphate buffer (pH 6.0) and kept in a constant temperature bath at the desired temperatures. 10-μl samples of enzyme solution were withdrawn at periodic intervals and cooled in an ice bath prior to the assay; the residual activity was measured and expressed as a percentage of initial activity. From a semilogarithmic plot of residual activity versus time, the inactivation rate constants (kr) were calculated (from the slopes), and apparent half-lives were estimated. Activation Energy Calculations—The thermal stability of GOD in the presence and absence of selected additives was determined by the inactivation rate constant (kr) as a function of temperature, in the range 56 – 67 °C. The temperature dependence of kr was analyzed from Arrhenius plot (natural logarithm of krversus reciprocal of the absolute temperature); the activation energy (Ea) was obtained from the slope of the plot. Activation enthalpy (ΔH*) was calculated according to the equation ΔH*=Ea−RT (Eq. 1) where R = universal gas constant, and T is the absolute temperature. The values for free energy of inactivation (ΔG*) at different temperatures were obtained from the equation ΔG*=−RTln(krh/kT)(Eq. 2) where h is the Planck constant and k is the Boltzmann constant. Activation entropy (ΔS*) was calculated from Equation 3. Δs*=(ΔH*−ΔG*)/T(Eq. 3) Effect of Lysozyme on Kmand Vmaxof GOD—Two kinetic parameters namely, the Michaelis-Menten constant (Km) and velocity maximum (Vmax) were calculated from the double reciprocal plot to study the effect of lysozyme on functional properties of GOD. The kinetics of GOD in 20 mm phosphate buffer (pH 6.0) was studied by varying the initial substrate concentration. Circular Dichroism Spectra Measurements—Circular dichroism measurements were made with a Jasco J-810 automatic recording spectropolarimeter fitted with a xenon lamp and calibrated with + d-10-camphor sulfonic acid. Dry nitrogen was purged continuously into the instrument before and during the experiment. The measurements were made at 30 °C (unless otherwise mentioned). The light path length of the cell used was 1 mm in the far-UV region, 5 mm in near-UV, and 10 mm in the visible regions. The protein concentrations were 0.2– 0.25, 0.7– 0.8, and 2.5–3.5 mg/ml in the far-UV, near-UV, and visible regions, respectively. The samples were prepared in 20 mm sodium phosphate buffer (pH 6.0). For the thermal unfolding measurements, data were collected at 222, 274, and 375 nm every second at a heating rate of 1 °C/min. The secondary structure of GOD was analyzed using the computer program of Yang et al. (17Yang J.T. Wu C.S. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Google Scholar), which calculates the structural component ratio of secondary structures for the protein, by the least squares method. The mean residue ellipticity [θ]MRW was calculated using a value of 115 for mean residue weight of GOD. Steady-state Fluorescence Measurements—Fluorescence measurements were made with a Shimadzu RF 5000 spectrofluorophotometer using a 10-mm path length quartz cell. GOD (1.5 μm concentration) in 20 mm phosphate buffer (pH 6.0) was used for measuring the intrinsic fluorescence. The temperature of the cell was maintained at 30 °C by circulating water through the thermostated cuvette holder. The emission spectra of intrinsic protein fluorescence were recorded after excitation at 285 nm. For ANS binding studies, an enzyme solution of 1.5 μm concentration was incubated with 20 μm ANS at 30 °C for 1 h, and spectra were recorded in the region 400 – 600 nm. The enzyme, in the presence of either 0.6 m NaCl or 0.2 m K2SO4, was incubated for 2 h at 30 °C before recording the spectra. Appropriate blank spectra of ANS in the corresponding salt concentrations were subtracted to obtain the fluorescence emission caused by ANS binding to protein. Size Exclusion Chromatography—Gel filtration measurements were carried out using a Shodex® PROTEIN KW-803 column (300 × 8 mm), with the manufacturer's exclusion limit of 1.5 × 105 for proteins, on a Waters HPLC system equipped with a 1525 binary pump and Waters 2996 photodiode array detector. For following the elution profile after thermal denaturation, both in the absence and presence of 0.2 m K2SO4, 20 μl of 4 –5 μm GOD solution was injected into the column at 30 °C before and after heating at 60 °C for 15 min. Elution of the sample was carried out isocratically using 20 mm phosphate buffer (pH 6.0) with a flow rate of 0.5 ml/min at 30 °C and detection at 280 and 375 nm by photodiode array detector. For Stokes radius measurements, the column was equilibrated with 20 mm phosphate buffer (pH 6.0), containing the desired salt concentrations, at 30 °C. 20 μl of 4 –5 μm GOD solution, equilibrated in the desired salt concentration (0 – 0.4 m K2SO4 in 20 mm phosphate buffer, pH 6.0) was injected into the column and eluted in the same buffer at 0.5 ml/min flow rate. The absorbance was detected at 280 and 375 nm. Standard proteins from a molecular weight marker kit for gel filtration (Sigma) including alcohol dehydrogenase (150,000), bovine serum albumin (66,000), carbonic anhydrase (29,000), cytochrome c (12,400) with known Stokes radius were used for calibrating the column. Blue dextran at a 1 mg·ml–1 concentration was used for determining the void volume. Experiments with Sulfhydryl Groups—The thiol groups exposed during the course of thermal unfolding of GOD were quantified by measuring their reactivity with DTNB as a function of temperature in a Gilford Response II spectrophotometer with an integrated temperature programmer. The transition was followed by increase in absorbance at 412 nm. The heating rate was 1 °C/min. The commercial preparation of GOD was purified by gel filtration on a column of Sephacryl S-200 HR. Homogeneity of the preparations was ascertained by HPLC and SDS-PAGE (Fig. 1). The purified enzyme had an absorbance ratio of 11.1 (280/450 nm), which is in good agreement with the reported value (4Tsuge H. Natsuakai O. Ohashi K. J. Biochem. 1975; 78: 835-843Google Scholar). Thermal Unfolding Transitions by Activity Measurements— GOD gets irreversibly inactivated over the temperature range 25– 80 °C. The residual activity of GOD as a function of temperature in the presence and absence of additives such as lysozyme, K2SO4, and NaCl is given in Fig. 2. These additives shifted the Tm of GOD from 59 °C to 61, 67, and 69 °C, respectively (Fig. 2). Thermal Inactivation Kinetics and Effect of Additives on the Thermal Stability of GOD—The thermal inactivation kinetics of native GOD was studied in the temperature range 56 – 67 °C in 20 mm phosphate buffer (pH 6.0). The effect of various additives, lysozyme, NaCl, and K2SO4, on the thermal stability of GOD, was followed by measuring the residual activity with time. At all temperatures studied, inactivation followed an exponential decay. The semilogarithmic plots (Fig. 3, A–D) indicated that thermal inactivation kinetics followed first order in all cases. The Arrhenius plots (Fig. 3Dinset) were linear in the temperature range studied. From this plot and making use of Equations 1, 2, 3, the activation parameters, free energy (ΔG*), enthalpy (ΔH*), and entropy (ΔS*) of activation, were calculated (Table I). The half-life of GOD was found to increase in the presence of each of the additives at all temperatures studied. Taking a typical case, at 60 °C, the half-life increased by 3.5-, 33.4-, and 23.7-fold, activation energy of GOD increased from 60.3 to 72.9, 76.1, and 88.3 kcal mol–1 whereas activation entropy increased from 104 to 142, 147, and 184 cal deg–1 mol–1 with 7.1 × 10–5m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The corresponding net free energy change, ΔG*, at 60 °C, was 0.9, 2.4, and 2.1 kcal mol–1, respectively. The magnitude of free energy of activation reflected the effectiveness of relative stabilization by various additives. The relatively small value of ΔG* (24.2 kcal·mol–1), for native GOD at 60 °C, pointed to the labile nature of enzyme. The difference in the slopes (activation energy) of Arrhenius plot (Fig. 3D, inset), in the presence of the lysozyme, NaCl, and the K2SO4 indicated the differences in mechanism of enzyme stabilization. A significant increase in the activation energy, Ea, in the presence of only 0.2 m K2SO4 (88.3 kcal·mol–1) compared with 1 m NaCl (76.1 kcal·mol–1) and 7.1 × 10–5m lysozyme (72.9 kcal·mol–1), indicated that stabilization of GOD by K2SO4 was of conformational origin. This was further confirmed by CD and size exclusion chromatography measurements. Stabilization of GOD (in terms of increased half-life and activation parameters) by NaCl and lysozyme in acidic pH values indicated the role of ionic interaction between GOD and lysozyme or NaCl. However, activation energy, i.e. the energy required to denature the enzyme, was higher in the presence of 0.2 m K2SO4 compared with either 1 m NaCl or 7.1 × 10–5m lysozyme. This indicated that hydrophobic interactions play a more dominant role in the stabilization of GOD than ionic interactions. The change in the activation entropy, ΔS*, in the presence of additives can be explained in terms of an enhancement of the order and compactness of the structure, thus favoring intramolecular stabilizing forces and consequently increasing the stability of the enzyme. Significant change in the activation entropy and the difference in the slopes of the Arrhenius plots in the presence of K2SO4 indicated that the stabilization of GOD was of conformational origin. CD measurements and size exclusion chromatography measurements confirmed this.Table IActivation parameters of GOD in the presence of additivesIncubation temperatureHalf-lifeInactivation rate constantΔG*ΔH*ΔS*°Cminkr × 10-4 s-1kcal·mol-1kcal·mol-1cal·deg—1·mol-1Native GOD (without additive) (Ea = 60.3 kcal·mol-1)56861.3425.259.5104.460139.624.259.5106.1637.51624.159.5105.5674.524.924.059.5104.3GOD in presence of 7.1 × 10-5 M lysozyme (Ea = 72.9 kcal·mol-1)563220.3626.072.3140.660462.525.172.3141.963245.024.873.3141.367129.524.972.3140GOD in presence of 1 M NaCl (Ea = 76.1 kcal·mol-1)5618060.06427.175.5146.8604340.2626.675.5146.8631460.7926.175.5147.067582.025.875.5146.2GOD in presence of 0.2 M K2SO4 (Ea = 88.3 kcal·mol-1)5614460.0827.087.6184.3603080.3926.387.6184.263621.925.587.61856727.54.325.287.6183.6 Open table in a new tab Effect of Lysozyme Concentration on the Stability of GOD— Detailed studies on the thermal inactivation of GOD were carried out in the presence of various concentrations of lysozyme at 60 °Cin20mm phosphate buffer (pH 6.0). As the mol ratio of lysozyme to GOD increased from 0 to 110, the half-life of GOD increased from 13 to 46 min (Fig. 4A), thereafter showing a decrease in the thermal stability of GOD. A significant observation (Fig. 4A) was that in the presence of higher concentrations of lysozyme (>150 mol ratio) the half-life of GOD decreased (seen as the dotted line). This was because of aggregation of lysozyme observed at higher concentrations. To avoid interference (of the aggregated lysozyme) during residual activity measurements, after exposing to 60 °C for the specified time, aggregated lysozyme was separated by centrifugation for 15 min at 4,000 rpm, and the supernatant was passed through a G-75 column (4 × 0.75 cm). The eluted sample was used to measure the residual activity of GOD. This procedure ensured that the half-life did not decrease after reaching the maximum. Increasing the lysozyme to GOD mol ratio above 110 gave no further improvement in the thermal stability of GOD, the lysozyme concentration corresponding to this mol ratio was employed for thermal inactivation studies. The requirement of a relatively high mol ratio (110) of lysozyme to stabilize GOD suggested that the interaction between lysozyme and GOD was nonspecific. Increased stability of GOD, in the presence of lysozyme in acidic pH (pH 6.0), confirmed the ionic interactions between lysozyme and GOD. At pH 7.7, the net charge on GOD was reported as –77 (18Voet J.G. Coe J. Epstein J. Matossian V. Shipley T. Biochemistry. 1981; 20: 7182-7185Google Scholar). The net charge (Lys and Arg) of lysozyme at pH 6.0 is positive (19Voet D. Voet J.G. Biochemistry. 2nd Ed. John Wiley & Sons, Inc., New York1995: 382Google Scholar). Effect of Lysozyme Incorporation on the Kinetic Parameters of GOD—To obtain a better understanding of the stabilization of GOD by lysozyme, kinetic parameters, Michaelis-Menten constant (Km) and maximum reaction velocity (Vmax), were determined in the presence as well as the absence of lysozyme (Fig. 4B). The Km and Vmax of GOD increased from 7.5 to 36.4 mm and 0.33 to 0.73 μm min–1, respectively, when the lysozyme concentration was varied from 0 to 1.1 mm. There was a decrease in affinity of GOD for β-d-glucose in the presence of lysozyme. Interaction of GOD with lysozyme resulted in an alteration of its functional properties. Thermal Inactivation of GOD: CD Measurements—The effect of thermal inactivation on the FAD environment, tertiary and secondary structures of GOD were studied by measuring CD spectra in the visible, near-, and far-UV regions, respectively. GOD exhibited characteristic FAD band at 375 nm in the visible region, a strong CD band at 274 nm in the near-UV region, and minima around 208 and 222 nm in the far-UV region. The analysis of secondary structure by the method of Yang et al. (17Yang J.T. Wu C.S. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Google Scholar) indicated 14% α-helix and 64% β-structure in the molecule. Because of the thermal inactivation of the enzyme, there were changes in all three regions of the spectra (Fig. 5, A–C). The intensity at 208 and 222 nm decreased, suggesting a loss in α-helix structure. The secondary structure analysis also supported this. The thermally inactivated enzyme had 9% α-helix content and 65% β-structure. In the near-UV region, the 274 nm band caused by the asymmetric environment of aromatic amino acids completely disappeared, indicating disruption of the native tertiary structure. The intensity of the CD band at 375 nm decreased, and the maxima was shifted to 335 nm. Addition of either 0.2 m K2SO4 or 0.6 m NaCl to the native enzyme did not affect the CD bands at 274 nm. In the far-UV region too, there was no change in the intensity of 222 nm band. Addition of either NaCl or K2SO4 did not alter the backbone or side chain conformation. Spectra of thermally inactivated enzyme at 60 °C in the presence of either 0.6 m NaCl (results not shown) or 0.2 m K2SO4 suggested (a) a small decrease in the intensity of both 274 nm band and 375 nm band, indicating the partial prevention of loss in tertiary structure and protection of the environment around FAD; (b) no significant change in the far-UV region, pointing to the prevention of loss of α-helix, attributable to these salts. The comparison of CD spectra of thermally inactivated GOD in the region 300 – 450 nm with that of free FAD suggested possible dissociation of FAD from GOD (Fig. 5C). Effect of Lysozyme, K2SO4, and NaCl on FAD Environment of GOD—To follow the conformational changes in GOD caused by interaction with lysozyme, the changes in CD spectra in the region 300 – 450 nm, where lysozyme does not contribute (even at high protein concentrations) were measured. The addition of lysozyme and NaCl resulted in small changes in the spectra, indicating a change in the environment of FAD in GOD (Fig. 6). K2SO4 did not affect the FAD band, indicating no significant changes in the environment of FAD. Thermal Unfolding Monitored by CD Measurements—The CD measurements of native and thermally inactivated GOD pointed to structural changes in the molecule. Secondary structural changes could be followed by changes in ellipticity values at 222 nm. Changes in tertiary structure were reflected at 274 nm, whereas the dissociation of FAD could be followed by changes in the ellipticity values at 375 nm. Thermal unfolding transitions of GOD in the temperature range 25–90 °C as followed at 222, 274, and 375 nm are shown (Fig. 7, A–C). The loss of tertiary and secondary structure in the native GOD, evident from [θ]274 nm and [θ]222 nm, occurred over a temperature range of 55 to 65 °C with a Tm of 62 °C. It was found that the loss of FAD (starting at 50 °C) was complete by 63 °C with a Tm of 59 °C. Effect of NaCl, K2SO4, and Lysozyme on Thermal Unfolding—To understand the mechanism of stabilization by NaCl and K2SO4, thermal unfolding transitions of GOD in the presence of 0.6 m NaCl and 0.2 m K2SO4 were followed by CD measurements at 222, 274, and 375 nm (Fig. 7, A–C). Tm, followed at 274 nm, shifted from 62 to 68 and 72 °C for native, 0.2 m K2SO4, and 0.6 m NaCl stabilized GOD, respectively (Fig. 7A). The Tm, followed at 375 nm, shifted from 59 °C to 68 and 72 °C for native, 0.2 m K2SO4, and 0.6 m NaCl, respectively. NaCl was seen to stabilize the tertiary structure, and the environment around FAD better compared with K2SO4. The only contrasting difference observed was the transition at 222 nm. K2SO4 was a marginally better stabilizer of the secondary structure compared with NaCl (Fig. 7B). Thus, it is evident that NaCl affected the side chain interactions (reflected by Tm measurements at 274 and 375 nm) more favorably, whereas K2SO4 primarily affected the backbone interactions. Tm followed at 375 nm, shifted by 7 °C in the presence of lysozyme (results not shown). Steady-state Fluorescence Measurements—For the native GOD, a fluorescence emission maximum was observed at 338 nm. The intrinsic fluorescence spectra of the holoenzyme in 20 mm phosphate buffer (pH 6.0), when excited at 285 nm, was significantly quenched compared with the heat-inactivated enzyme (Fig. 8A). For the heat-inactivated enzyme, a significant enhancement of fluorescence intensity along with a small shift in the emission maximum was observed. The dissociation of FAD from the enzyme because of heat inactivation resulted in an increase in the quantum yield. Studies on the reduced and oxidized holoenzyme as well as the apoenzyme revealed that in the native conformation of the enzyme, seven tryptophan residues and FAD are in proximity. The quenching of fluorescence was the result of a Förster energy transfer from the tryptophan residues to the flavin group (20Haouz A. Twist C. Zentz C. Kersabiec A.M. Pin S.