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 UV-
VIS 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 pH influence on bovine liver catalase structure by means

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
of EPR and UV-VIS spectroscopy.
Experimental procedures
Polycrystalline bovine liver catalase and spin label 3-(2-
Iodoacetamido)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–

3
pH7
Absorbance [a.u.]

pH10
2 pH2
a)
1

0
220 240 260 280 300 320
0,3
Wavelength [nm]
Absorbance [a.u.]

b)
0,2

0,1

340 360 380 400 420 440

0,08
Wavelength [nm]
Absorbance [a.u.]

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. Brunelli L., Yermilow V., Beckman J.S.: Modulation
of catalase peroxidatic and catalatic activity by nitric
oxide. Free Radical Biology & Medicin 30(7), 709-
714, 2001.
2. 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.
3. 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.
4. Tarhan L.: Use of immobilized catalase to remove
H2O2 in the sterilization of milk. Process Bioche-
mistry 30, 623-628, 1995.
5. 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.
6. Abraham B.D., Sono M., Boutaud O., Shriner A.,
Dawson J.H., Brash A.R., Gaffney B.J.: Charac-
terization of the coral allene oxide synthase active site
with magnetic circular dichroism and electron para-
magnetic resonance spectroscopy: evidence for tyrosi-
nate-ligation to the ferric enzyme heme. Biochemistry,
40, 2251-2259, 2001.
7. Bartoszek M., KĞciuczyk M.: Study of the temperature
influence on catalase using spin labelling method. J.
Mol. Struct.,744-747, 733-736, 2005.
8. 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.
9. 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.