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BIOLOGIA P L A N T A R U M (PRAHA)

1 ( 1 ) : 3 - - 8 , 1959

Catalase Activity in Thermal Blue-green Al9ae in Relation


to Temperature

~T]~PA.N K U B : [ N

Department of Plant Physiology, Biological Faculty of Charles


University, Praha. The Botanical Garden of the Slovak Academy of Sciences, Kohi(.e

Received April 24. 1957

Souhrn

Autor sledoval aktivitu katalasy terms sinic v zAvislosti na tcplot~. Jako


materiAlu bylo pou~ito homogenAtu i termgdnich sinic r. Oscillatoria (teplotnl op-
timum 38 ~ C). ManometrickA metoda podle APPLEMANA (1951) byla ponSkud
modifikovAna, ale princip (stanoveni uvoln~n~ho kysllku v prvnich fs reakce)
zflstal zachovs Na rozdil od b ~ n ~ c h manometrick:~ch m e t e d se b~hem stanoveni
nezachovs konstantnl objem (Vo) a registruje se 5as pot~ebn:~ ke zm~n~ tlaku
o urSit:~ interval. V~sledky se p~epoSitAvaji na V o graficky.
Nebyly zji~t~ny ~hdn~ zvlAhtnl vlastnosti katalasy terms sinic, pokud jde
o zAvislost jeji aktivity na teplot~, co~ potvrzuje p~edpoklad M. B. ALLENOV]~
(1950), ~e neexistuji zvlg~tni bilkoviny resistentni k vysok:~m teplotAm. ReakSni
rychlost se v zAvislosti na teplot5 zvy~uje a~ do 55 ~ C. PH teplot~ 60 ~ C je enzym
z tormAlnich sinic prakticky kvantitativn~ denaturovAn, stejn~ jako katalasa
z mesofilnich mo~sk:~ch ~as nebo z b:~Sich jater.

Summary
The author has studied the relation of the catalase activity in thermal
blue-green algae to temperature. Experimental data were taken from the
first phase of the reaction, so that oxidation of the enzyme b y the substrate
was eliminated. The relation of the activity of a homogenate of thermal blue-
green algae to temperature gives a curve similar to that obtained b y SIZER
(1944) for pure enzyme preparations isolated from beef livers. The resistance
of the enzyme to high temperatures is the same in thermal blue-green algae
as in mesopholic seaweeds. The catalase of thermal blue-green algae does
not exhibit any special characteristics differing from the catalase of mesophiles.

Introduction
The thermophily of some organisms still remains incompletely explained,
although we know b y now for certain that their metabolism is essentially
the same as that of mesophiles, that no natural division with regard to temper-
ature exists between thermophiles and mesophfles and that thermophily is
a characteristic which organisms of the same species, can gain and lose (Sporo-
vibrio desul/uricans ).
4 ~. KUBIN

On the basis of her experiments ALLEN (1950) has come to the conclusion
that the nature of thermophily is not to be sought in the specific resistance
of plasmatic proteins to high temperatures. In her opinion the more intense
metabolism at high temperatures is responsible for the higher resistance of
thermophilic organisms, the synthesis of their enzymes and other plasmatic
components taking place more rapidly than their denaturation. This assump-
tion has been based on the direct relationship between consumption of nutri-
ents from the environment and rising temperature. With insufficient nutrients
thermophilic species or strains (Bacteria) perish at high temperatures as
quickly as mesophilic species.
Blue-green algae are characterised by the wide temperature limits within
which they can exist and the thermophily of many species can be regarded
rather as facultative than strict. In connection with the study of the tempera-
ture dependence of photosynthesis and respiration in blue-green algae the
author also observed the dependence of catalase activity on temperature.

Materials and methods

T h e r m a l blue-green algae of the genus Oscillatoria (with o p t i m u m t e m p e r a t u r e for g r o w t h ,


p h o t o s y n t h e s i s a n d respiration a t t e m p e r a t u r e s close to 35 ~ C). B o t h intact algae filaments a n d
ilomogenate were used. A pure 14-days-old culture of the species chosen cultivated in a small
R o u x flask (20 ml. m e d i u m ) was homogenised for 3 m i n u t e s in a n all-glass homogeniser after
POTTER a n d ELVEHJEbl (1936) w i t h 10 ml. p h o s p h a t e buffer p H = 7 (Na2HPO 4 M/15 + K H ~ P O 4
M/15). I n each case 2.7 ml. of this suspension, corresponding to 0.25 rag. of total nitrogen, was
p i p e t t e d into a m a n o m e t r i c flask. B y using cultures f r o m eight cultivation dishes e n o u g h ma-
terial w a s o b t a i n e d for all tests. By preliminary e x p e r i m e n t s it was established t h a t the catalase
activity of the h o m o g e n a t e was n o t changed even after 24 tmurs if kept at 0 ~ C in the dark.
This period sufficed for the d e t e r m i n a t i o n of enzyme activity at all t e m p e r a t u r e s chosen w i t h
a sufficient n u m b e r of repetitions.
I n d e t e r m i n i n g the t e m p t e r a t u r e coefficient of enzymatic splitting the c o n c e n t r a t i o n of the
s u b s t r a t e m u s t be chosen w i t h a view to the c o n c e n t r a t i o n of the enzyme so t h a t t h e time de-
pendence of the rate of reaction should be suited to the kinetic e q u a t i o n for " z e r o - o r d e r " reactions
w h i c h are characteristic for heterogenous catalysis (FRUTO~r a n d StM~ONDS 1954). F o r this r e a s o n
in the p r e l i m i n a r y e x p e r i m e n t s h y d r o g e n peroxide was added to the suspension of h o m o g e n i s e d
algae to give r e s u l t a n t concentrations of 0.033 M, 0.1 M a n d 0"3 M. P r e l i m i n a r y d e t e r m i n a t i o n s
were carried o u t at a t e m p e r a t u r e of 20 ~ C. The a m o u n t of o x y g e n evolved in relation to time agreed
i n the time interval used with the kinetic e q u a t i o n of " z e r o - o r d e r " r e a c t m n s

- - dC/dt ~ k E

(where E is the a m o u n t of enzyme present) only w i t h a r e s u l t a n t c o n c e n t r a t i o n of the s u b s t r a t e


of 0-1 M. I n this a n d all o t h e r e x p e r i m e n t s the material w a s incubated at the given t e m p e r a t u r e
for 10 m i n u t e s . The c o n c e n t r a t i o n 0.3 M already caused a m a r k e d d e n a t u r a t i o n of the enzyme,
while a t the c o n c e n t r a t i o n of 0.033 M H20 ~ the reaction took place clearly in accordance w i t h
the kinetic e q u a t i o n for a m o n o m o l e e u l a r reaction

- - dC/dt = kC

I n view of the s t r o n g l y oxidising properties of the s u b s t r a t e it is n o t possible in the last case


to decide to w h a t e x t e n t the decrease in reaction rate in the course of time in the last phase is
related to the c o n c e n t r a t i o n of the s u b s t r a t e or to the d e n a t u r a t i o n of the orlzyme. Therefore, in
f u r t h e r experiments, a r e s u l t a n t c o n c e n t r a t i o n of 0.1 M a n d a s o m e w h a t modified m e t h o d after
APPLEMAN (1951) were 'used which m a d e it possible to determine rate c o n s t a n t d u r i n g the first
t h i r t y seconds following t h e s t a r t o f the reaction. Manometric .determination f o r m s the basis
of this m e t h o d , b u t the time corresponding to the change in pressure in a given interval is recorded.
CATALASE ACTIVITY IN T H E R M A L B L U E - G R E E N ALGAE 5

W i t h the e x p e r i m e n t s described this interval was 10 m m . of m a n o m e t r i c liquid. The rate of re-


action would m a k e it difficult to m a i n t a i n the v o l u m e of gas Vg a t a c o n s t a n t level. I n order to
avoid this difficulty the values o b t a i n e d were calculated acSording to the m a t h e m a t i c a l l y
derived a n d e x p e r i m e n t a l l y confirmed relation

P. Vm
h = 2h 1 + - - ,
Vg

where 2h 1 is the change in pressure in m m . of m a n o m e t r i c liquid w h e n the v o l u m e of gas Vg


is n o t m a i n t a i n e d a t a c o n s t a n t level (h 1 is the change in level in one a r m of the m a n o m e t e r ) a n d
V m is the v o l u m e of gas f o r m e d in the capillary above the liquid in t h e closed a r m of the mano-
meter. The r e m a i n i n g s y m b o l s are c o m m o n in m a n o m e t r y .
F o r the p u r p o s e followed W a r b u r g ' s r e s p i r o m e t e r of usual t y p e w i t h a vessel lacking the central
c u p a n d w i t h one side a r m w a s used.
The above relation is only a p p r o x i m a t e , as follows f r o m its derivation:

p V = (P + h ) . V g (1)
pV = (P + 2h~). ( V g + Vm) (2)

B y c o m p a r i s o n of the right sides of these e q u a t i o n s the following expression is o b t a i n e d

Vg -[- V m P . Vm
h = 2h 1 -- -~ - -
Vg Vg
Since
Vg-~- V m ~ V g ,
then
Vg + V m
Vgo --
a n d therefore this expression can be ignored in the formula. W i t h i n t h e pressure range of the
W a r b u r g m a n o m e t e r it is possible in m o s t cases to ignore the error (less t h a n 30/o) so produced.
F o r example, if Vg = 16,850 1., t t h l ~ 300 ram. a n d V m ~ 800/tl. (the v o l u m e of gas correspond-
ing to 1 ram. of m a n o m e t r i c liquid m u s t be k n o w n ; in o u r case it is 2.67 td.) Calculating h
according to the original f o r m u l a the r e s u l t a n t value is 1105 ram., w h e n using the simplified
f o r m u l a h is 1075 ram.
W i t h i n the pressure range of the W a r b u r g m a n o m e t e r the relation b e t w e e n h a n d h 1 is also
practically linear, w h i c h can be usefully e m p l o y e d in the graphic evaluation of the results.
Time intervals corresponding to a change in pressure of 10 ram. of m a n o m e t r i c liquid were
registered photographically. A spring m a c h i n e was used in which a c o n s t a n t speed was ensured

Fig. l. P h o t o g r a p h of original time records f r o m which the rate of decomposition of h y d r o g e n


peroxide by the catalase was worked out.
A) 0 ~ B) 30 ~ C) 50 ~
6 ~. K U B f X

b y a centrifugal regulator. To the machine there was a t t a c h e d a d r u m w i t h grooves for the in-
sertion of polarographic p a p e r a n d w i t h a signalling a p p a r a t u s to a n n o u n c e the beginning a n d
the end of the record paper. The speed of r o t a t i o n w a s one t u r n in t w o a n d a h a l f m i n u t e s (r
= 8 cm.). Opposite the d r u m there w a s a slit w i t h t w o signal electric b u l b s one above the other.
The c o n t a c t s of m e t r o n o m e were connected to the circuit of one b u l b recording the interval of
one second. The circuit of the other b u l b was connected b y the pressing of a switch at the m o m e n t

I ! I !

300 ' i
I
/-
I

/
250 0,8

200
,.; 0,6
-,t,
0///
/0
o

:L
100

q2
5C

G I | I | I
0 20 40 60 80 sec. 0 20 40 6O
~
Fig. 2. R a t e of liberation of o x y g e n f r o m Fig. 3. Relation of the catalytic activity of
0.1 M solution of h y d r o g e n peroxide a t va- the Oscillatoria h o m o g e n a t e to t e m p e r a t u r e .
rious t e m p e r a t u r e s . The reaction w a s catal- The m e a s u r e of activity of the h o m o g e n a t e
ysed b y a h o m o g e n a t e of the alga Oscilla. is the original rate of decomposition of h y d r o -
toria at pI-I = 7. gen peroxide expressed in micromols of I~O~
distributed in seconds.

w h e n a change in pressure of 10 m m . of m a n o m e t r i c liquid t o o k place. I n this way, after developing


the records, t w o rows of signs were obtained, of which one w a s a m e a s u r e of the time in seconds
a n d the o t h e r i n d i c a t e d - - a c c o r d i n g to this s c a l e - - t h e time necessary for a change in p r e s s u r e
of the degree chosen to occur (fig. 1). I t is possible to read the pressure w i t h sufficient a c c u r a c y
e v e n d u r i n g shaking (100 oscillations to the minute). I t is best to read it a t the m o m e n t w h e n
t h e m o v i n g m e n i s c u s first reaches each fifth g r a d u a t i o n of the scale.

Results

The course of the decomposition of hydrogen peroxide in relation to time


is given in fig. 2. The relation of the rate constant determined (M H~O~/sec.)
to temperature is illustrated in fig. 3. Deviations from the theoretically pre-
CATALASE ACTIVITY IN T H E R M A L B L U E - G R E E N ALGAE 7

sumed exponential curve cannot be fully explained since the work was not
carried out with the pure enzyme but with a suspension of homogenised mate-
rial. Therefore, not even the logarithmic expression of the constant in relation
to 1/T gives a straight line (fig. 4).
Activation energies for individual temperature intervals were calculated from
Arrhenius's equation ~ 2-303 9 R logI/T1
k 2 -__
- l ol/T2
g k~ (where I~ is the gas constant
expressed as 1.987 cal./mol.). They
are set out in the following table: o.5
0 to 50 ~ C 4100 cal.
0 to 10 ~ C 5500 cal. t ~
10 to 40 ~ C 2900 cal. O0
40 to 50 ~ C 6400 cal.
50 to 60 ~ C 59,000 cal.

Experiments with whole, non-


homogenised material gave similar
results. For obvious reasons their o.2 (-, = 5 9 0 0 0 " ~'o
dispersion was, however, rather
greater.
The results obtained do not show Oa~ ! I I | ! I

any essential differences from these 3,0 3.2 3,~ 3,6


of SIZER (1944), using catalase iso- l/T.fo a
lated from beef liver, or from those
of the Japanese author TAKAGI Fig. 4. Relation of the l o g a r i t h m of the rate
c o n s t a n t of decomposition of h y d r o g e n perGxide
(1953) who worked with five spe- (catalysed b y h o m o g e n a t e of the alga Oscillatoria)
cies of seaweeds: Ulva pertusa, to the inversion value of absolute t e m p e r a t u r e at
Enteromorpha linza var. crispata, which the reaction took place.
Scytosiphon lomentaria, Polysiphonia
venticulosa and Porphyra ?seudolinearis. As far as the resistance of the
catalase to high temperatures is concerned, the results are also numerically
in complete agreement both for marine mcsophiles and for thermal blue-
green algae. BONICHSEN and co-workers (1947) obtained, on the basis of
their experiments with very pure preparation, a considerably lower value of
activation energy (1700 cal.) and they stated t h a t higher values were obtained
only when working with imperfectly pure preparations. For denaturation
SrZER has quoted a lower activation energy (51,000 cal.), but this can
be explained by differences in method (different incubation period, etc.).
I t seems, then, t h a t the catalase of thermal blue-green algae does not exhibit
any special characteristics as regards the relation of its activity to temperature
and resistance to high temperatures.

References

ALLEN, M. B.: The d y n a m i c n a t u r e of t h e r m o p h i l y . - - J. Gem Physiol. 33 : 205--210, 1950.


APPLEMAN, D.: Manometric d e t e r m i n a t i o n of catalase activity. A p p a r a t u s a n d m e t h o d . - - Anal.
Chem. 23 (11) : 1627--1631, 1951.
BONNICHSEN, R. K., CHANCY],B. a n d THEORELL, H.: Catala%o activity. Acta chem. Scand.
I : 685--709, 1947.
8 ~. K U B I N

FRv~o~, J. S., Si~O~DS, S.: General Biochemistry. (pp. 235--260). New York, 1954.
POTTER, V. 1~., ELVEH~EYl, C. A.: (cit. after Kleinzeller, A., MAlek, J., Vrba, R.: Manometrick6
motherly a jejich pou~iti v biologii a biochemii. Praha, 1954): Manometric methods and their
application in biology and biochemistry. - - J. biol. Chem. 114 : 495--504, 1936.
SIZER, I. W.: Temperature activation and inactivation of the crystalline catalase-hydrogen
peroxide system. - - J. biol. Chem. 154 : 461--473, 1944.
TAKAGI, M.: On the stability against heat of the catalase in marine algae. - - Bull. Jap. Soc. Sci.
Fish. 19 (8) : 886--888, 1953.

Address: ~t~pAn Kubin, prom. biol., Botanical Garden of the Slovak Academy of Sciences,
Komcnsk~ho 67, Ko~ice, Czechoslovakia.

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