EFFECTS OF RADIATION ON LACTATE
DEHYDROGENASE
A. Hategan, D.Martin, C. Butan, V.V.Morariu*
Institute of Atomic Physics, Electron Accelerator Laboratory, P.O.Box MG-36,
R-76900,Magurele, Bucharest, Romania, e-mail: [email protected]
*
Institute of Isotopic and Molecular Technology, P.O. Box 700, 3400
Cluj-Napoca, Romania
ABSTRACT
Results on the influence of 6 MeV electron beam and 2.45 GHz 565 W microwaves as
well as of the combined treatement, at -3 o C and -196 o C, on lactate dehydrogenase activity
are presented. Our data showed a moderate inactivation of the electron irradiated lactate
dehydrogenase, in the dose range (0-4.2 kGy). The microwave irradiated samples exhibited a
non-linear behaviour (successive activation and inactivation of the enzyme molecules)
suggesting the major influence of the nonthermal component of microwave radiation. The
combined irradiation lead to similar behaviour of the enzyme as in the case of electron
irradiation.
Key-words: lactate-dehydrogenase, electron irradiation, microwaves.
INTRODUCTION
There is little information on enzyme inactivation with high LET radiations, and
electron irradiation was used only in few studies /1,2,3,4/ and still the molecular mechanisms
induced are not completely clarified /5,6/. Freezing the samples at low temperatures makes
possible the determination of global non-thermal effects (specific effects induced by
electromagnetic radiation interaction with the biosystem, distinct from the changes induced by
heating that occurs in the irradiated samples/6/) of microwaves/6,7,8/ and the direct effects of
electron irradiation (due to direct interaction radiation-biomacromolecule)/5/.
The aim of this paper is to study the direct effects of 6 MeV electron beam and 2.45
GHz microwaves, at -5 o C and -196 o C irradiation temperatures,on lactate dehydrogenase.
.METHODS
Crystallised lactate dehidrogenase in ammonium sulphate solution 3.2 M, pH aprox. 6
was diluted in the ratio 5 l crystallised enzime/3 ml bidistilled water.. 1 ml aliquots of the
suspension were placed in thermoresistant plastic trays , for irradiation procedure. The
samples were frozen at -3 oC using an ethanol-ice bath, and at -196 o C by: a) rapidly
immersing the samples in liquid nitrogen, b) freezing the samples at -3 o C (using an
ethanol-ice bath) or c) at -10 o C (using a methanol-ice bath) and afterwards immersing them
in a liquid nitrogen bath.They were irradiated in a rectangular cavity designed to permit either
electron beam irradiation only, or microwave irradiation only or simultaneous electron beam
and microwave irradiation. A microwave generator with the power op to 850 W at 2.45 GHz
is coupled to the rectangular cavity sidewalls by means of a floated waveguide system.
1
Scanned electon beam from the ALIN 10 linac of 6 MeV (average intensity of 5-10 A,
frequency of the pulses 100 MHz, time of a pulse 5-10 s) was introduced perpendicularly by
the upper wall end of the thickness of 0.1 mm.y The monitor of the ionising chamber situated
under the samples was calibrated in dose units using Fricke dosimetry before irradiation and
the dose rate was estimated to be constant during irradiation.
Lactate dehydrogenase catalyses the hydrogen transfer reaction:
piruvate + NADH + H+ <----------> lactate + NAD+
The enzymatic activity of lactate dehidrogenase was determined by measuring the rate
of extinction decrease at 340 nm of NADH accompanied by the reduction of pyruvate to
lactate, according to the technique of Bergmeyer et al./9/.
RESULTS AND CONCLUSIONS
Lactate dehydrogenase presents partial unfolding when freezing and thawing /10/, that
leads to an irreversible loss of the enzymatic activity. The optimal procedure to prepare the
frozen enzyme suspesions for irradiation seemed to be procedure c,which reduces the activity
only to 82 %.
It is known that radiation inactivation of enzymes is mainly monoexponential with
increasig the dose /1,5,11/. The total inactivation of frozen enzymes, due to direct effects of
radiation is obtained for large electron doses as 10-50 kGy /5/. We choosed the electron dose
range and irradiation time periods (the same for both radiations) so that the samples to remain
in frozen state during microwave irradiation.
High energy electron irradiation of frozen lactate dehydrogenase at -3 o C and -196 o C
, independent on the procedure of freezing in the dose range 0-350Gy and 0-4.2 kGy leads
to a insignificant respectivelly moderate decrease (mainly down to 50% of the frozen enzyme
activity) of the enzymatic activity (figures 1-3).
Microwave irradiation leads to variations of the enzymatic activity, presenting zones
of activation of the enzyme molecules followed by zones of drastic decrease.(figures 1-3),
suggesting the major influence of the nonthermal component of microwave radiation. The
most semnificative variations are presented by the samples irradiated at -3 o C.(figure 1). The
activation of enzyme molecules might be explained by radiation action on the inactive enzyme
molecules present in the suspension ( obtained inevitably by the standard procedure of enzyme
separation from the tissues, and by freezing). Similar repeated results were obtained also for
400 W and 495W microwave powers,but not for 310W microwaves (data not presented here),
indicating that the activation of frozen enzyme molecules takes place only at large power
levels, the bigger the power level, the increased activation being obtained.
The combined irradiation lead to similar behaviour of the enzyme as in the case of
electron irradiation.
Our data inform on electron beam and microwave radiations influence on the
enzymatic activity loss during freeze-thawing, when the subsequent growth of the water
crystals influences the two and three dimensional structure of the enzyme /10/. The system
studied is very complex because of the superposition of freeze-thawing effects on the
irradiation effects, both molecular mechanisms being incompletely clarified yet. Further
studies are needed to clarify both aspects.
2
Enzimatic Activity(%)
100
electrons
microwaves
electrons+microwaves
80
60
40
20
0
20
40
60
80
100
120
Irradiation Time(s)
1.Effects of radiation on lactate dehydrogenase activity. The enzyme was frozen at –3o C
before irradiation. Electron irradiation was performed at dose rate of 11 Gy/s.
2.Effects of radiation on lactate dehydrogenase activity. The enzyme was frozen at–196o C
(two step technique: -3o C, -196o C) before irradiation. Electron irradiation was performed at
dose rate of 43 Gy/s.
Irradiation Time(s)
0
5
10
15
20
25
30
35
)%(ytivitcA citamiznE
0
50
100
electrons+microwaves
microwaves
electrons
150
200
3
40
Enzimatic Activity(%)
200
electrons
microwaves
electrons+microwaves
150
100
50
0
20
40
60
80
100
Irradiation Time(s)
3.Effects of radiation on lactate dehydrogenase activity. The enzyme was frozen at –196o C
(two step technique: -3o C, -196o C) before irradiation. Electron irradiation was performed at
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