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Polish J. of Environ. Stud. Vol. 15 No. 4A (2006), 41-43 The Study of pH Influence on Bovine Liver Catalase by Means of UV-VIS Spectroscopy and Spin Labelling Method M. Bartoszek, W. W. Suákowski Institute of Chemistry, Department of Chemistry and Environmental Technology, University of Silesia, Szkolna 9, 40-006 Katowice, Poland Abstract The EPR and UV-VIS spectroscopy was used to determine the pH effect on bovine liver catalase. The measurements were made in the pH range from 2 to 10. The positions, shapes and relative intensities of the UVVIS peaks were observed. Spin labelling technique was used to monitor the molecular dynamics of catalase. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the haeme group from the protein. The decrease of respective absorption bands and their shifts under acid and alkaline pH correlate well with the changes of rotational correlation time and w/s parameter. Keywords: catalase, denaturation, dissociation, spin labelling, UV-VIS spectroscopy Introduction Oxidative metabolism often results in the generation of active oxygen intermediates, which must be neutralized in order to minimize collateral damage to the organism. Haeme-containing catalase is the source of protection by scavenging hydrogen peroxide, decomposing H2O2 to O2 and H2O. It is implicated in ethanol, methanol, formate and azide decomposition [1], inflammation, apoptosis, aging and cancer [2]. However, catalase could be potentially applied for degradation of residual peroxide in textile bleaching baths, thereby providing water and energy savings [3] and to remove H2O2 used in sterilization of milk [4]. The pH of environment has a considerable influence on the activity of any enzyme. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. The value of pH optimum for catalase is 7.0 [3-6]. However, it is known that activity of catalase does not change in the pH range 5.1 – 8 [3, 4] and below pH 3.0 acid denaturation is observed [5]. The objective of this work was to study the pH influence on bovine liver catalase structure by means of EPR and UV-VIS spectroscopy. Experimental procedures Polycrystalline bovine liver catalase and spin label 3-(2Iodoacetamido)PROXYL (IAA) was purchased from Sigma Aldrich. Buffer solutions (pH = 2, 4, 6, 7, 8, 10) were purchased from POCH Gliwice. The method of spin labelling of catalase was described previously [7]. Electron paramagnetic resonance (EPR) spectra were obtained with Bruker EMX EPR spectrometer at the X-band (9 GHz) at the room temperature. Spectral properties of bovine liver catalase were monitored by recording UV/VIS absorption with Perkin Elmer Lambda Bio spectrophotometer. All measurements were performed triplicate. Results and discussion Absorption spectrum obtained for catalase in the range of O = 220-330 nm on the Fig.1a is presented. This band 42 Bartoszek M., Suákowski W. W. originates from aromatic aminoacids. The changes observed in this region give evidence of rearrangement in the structure of globule. The intensity of this band is a measure of the content of helical conformation in polypeptide chains and shows progress of the induced denaturation process. Along with pH change a slight reduce of absorbance is observed, indicating conformational changes in molecule. The most visible changes in the Soret band (350 – 450 nm) are observed (Fig. 1b), giving evidence about haeme group degradation. This band is connected with the presence of haeme in each catalase monomer. It arise from S-S* transitions in the haeme system. The Soret band is very sensitive to variation of the microenvironments around the prosthetic group. Previous studies have shown that this band will change or diminish if the protein is partially or fully denatured [5,6]. The removal of the haeme group and degradation of molecule is observed especially in acidic environment, where both decrease of absorbance (from A = 0,23 for pH 7 to A = 0,16 for pH 2) and shift toward shortest wavelength (from O = 405 nm for pH 7 to O = 382 nm for pH 2) is observed (Fig 1b). These facts suggest a breakage of some of the haeme-protein bonds present in the native enzyme. It has been reported on the basis of magnetic susceptibility measurements that the sqare of the effective Bohr magneton decreased at a given acidic pH and increased at a given alkaline pH [8]. This fact indicate that acidic environment causes the spin-state change in iron ion contained in heme group from the high (S = 5/2) to the low (S = 1/2), while alkaline just the opposite (from the low to the high spin-state). Thus, in the case of acidic environmental the length of Fe-N bound in porphyrine is shorter than in alkaline pH, what the access of substrate to the active site makes difficult. In the wavelength range 450– Absorbance [a.u.] 3 pH7 pH10 pH2 2 a) 1 Absorbance [a.u.] 0 220 0,3 260 280 300 320 Wavelength [nm] b) 0,2 0,1 0,08 Absorbance [a.u.] 240 340 360 380 400 420 440 Wavelength [nm] 0,06 c) 0,04 0,02 450 500 550 600 650 700 Wavelength [nm] Fig. 1. The effect of pH on UV-VIS spectrum of catalase in the wavelength range: a) 220-330 nm, b) 330-450 nm, c) 450-700 nm The Study of pH Influence… 750 nm in native enzyme 3 characteristic maxima are observed: 504, 538 and 640 nm (Fig. 1c). The 504 and 640 nm peaks are high-spin charge-transfer porphyrin (pS) to iron (dS) bands, 538 nm peak originated from S-S* transitions in the haeme group [6]. Both acidic and alkaline environment causes decrease of absorption bands and the shift of peaks. The changes are visible especially when we observe charge transfer band (O § 630 nm). Both acidic and alkaline pH cause decrease of absorption bands and significant shift of absorption peaks towards longer wavelength. The changes are observed above pH 8 and below pH 6. The UV/VIS spectroscopy findings are consistent with the results observed by use of spin labelling method. The main parameter, giving information about spin labels located in molecule is correlation time Wc [7]. According to expectation the highest correlation time value is obtained when pH 7 (Wc = 3.51*10-10). However, the change of pH both toward acidic and alkaline environment causes decrease of correlation time value (Wc = 1.21*10-10 for pH 2, Wc = 1.84*10-10 for pH 4, Wc = 3.41*10-10 for pH 6, Wc = 3.11*10-10 for pH 8, Wc = 2.13*10-10 for pH 10). The decrease of the correlation time to about 40% of the Wc obtained for native catalase indicates denaturation of enzyme. It can be concluded that below pH 4 acid denaturation of catalase takes place. It has been reported that alkaline denaturation occurs over pH 12 [9]. The results obtained in the range pH 7 – 10 show the changes in the quaternary and tertiary structure of catalase, but up to pH 10 complete denaturation is not observed. The decrease of correlation time is consistent with the increase of W/S parameter. This parameter is the ratio of the spectral amplitude of the low-field weakly immobilized line (W) to low-field strongly immobilized line (S). The W/S value increases up to pH 7 and than decreases in the range pH 7 to pH 10 (W/S = 31.8 for pH 2, W/S = 18.08 for pH 4, W/S = 12.34 for pH 6, W/S = 11.98 for pH 7, W/S = 14.14 for pH 8, W/S = 15.61 for pH 10). As the pH drops into the acidic range the enzyme tends to gain hydrogen ions from the solution. As the pH moves into the basic range the enzyme tends to lose hydrogen ions to the solution. In both cases the decrease of enzyme activity is observed. Conclusions Treatment with acid or alkaline solutions causes changes of UV-VIS spectra which may be due to dissociation of the enzyme into subunits and partial or 43 complete removal of the haeme group from the protein. Below pH 4 acidic denaturation takes place. However up to pH 10 complete alkaline denaturation is not observed. The changes of absorbance, correlation time and W/S parameter value are coherent and exhibit variation in the quaternary and tertiary structure of catalase, dissociation of tetramer into subunits and refolding of polypeptide chains. References 1. 2. 3. 4. 5. 6. 7. 8. 9. Brunelli L., Yermilow V., Beckman J.S.: Modulation of catalase peroxidatic and catalatic activity by nitric oxide. Free Radical Biology & Medicin 30(7), 709714, 2001. Putnam Ch.D., Arvai A.S., Bourne Y., Tainer J.A.: Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism. J. Mol Biol. 296, 295-309, 2000. Costa S.Z., Tzanov T., Carneiro A.F., Paar A., Gubitz G.M., Paulo A.C.: Studies of stabilization of native catalase using additives. Enzyme Microb. Technol. 30, 387–391, 2002. Tarhan L.: Use of immobilized catalase to remove H2O2 in the sterilization of milk. Process Biochemistry 30, 623-628, 1995. Samejima T., Mijahara T., Takeda A., Hachimori A., Hirano K.: On the acid denaturation of porcine erythrocyte catalase in relation to its subunit structure. J. Biochem (Tokyo) 89(4), 1325-32, 1981. Abraham B.D., Sono M., Boutaud O., Shriner A., Dawson J.H., Brash A.R., Gaffney B.J.: Characterization of the coral allene oxide synthase active site with magnetic circular dichroism and electron paramagnetic resonance spectroscopy: evidence for tyrosinate-ligation to the ferric enzyme heme. Biochemistry, 40, 2251-2259, 2001. Bartoszek M., KĞciuczyk M.: Study of the temperature influence on catalase using spin labelling method. J. Mol. Struct.,744-747, 733-736, 2005. Yoshida K., Iizuka T., Ogura Y.: Effect of pH on the optical absorption spectrum and effective number of the Bohr magneton of horse erythrocyte catalase In the range from 77K to 267K. J. Biochem. 68, 849-857,1970. Takeda A., Hirano K., Shiroya Y., Samejima T.: On the denaturation of porcine erythrocyte catalase with alkali, urea, and guanidine hydrochloride in relation to its subunit structure. J Biochem (Tokyo) 93(4), 967-75, 1983.