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FISHERIES RESEARCH BOARD OF CANADA
Translation Series No , 568
1
Thermal denaturation of enzyme proteins
of cold-blooded animals
By G. Siebert, A. Schmitt, R. v. Malortie
and E. Adloff
Thermische Denaturierung von
Original title
Kaltbliiter-Enzymproteinen
Fromz Experientia, Vol. 16, p. 491, 1960.
Translated by Elisabeth Czeija, Bureau for
Translations, Foreign Language Division,
Department of the Secretary of State of Canada
Fisheries Research Board of Canada
Technological Research Laboratory,
Halifax, N. S.
1965
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DEPARTMENT OF THE SECRETARY OF STATE.'
BUREAU FOR TRANSLATIONS
SECRÉTARIAT D'ÉTAT
BUREAU DES TRADUCTIONS
FOREIGN LANGUAGES
DIVISION DES LANGUES
ÉTRANGÈRES
DIVISION
INTO —
TRANSLATED FROM — TRADUCTION DE
ENGLISH
GERMAN
SUBJECT — SUJET
Fisheries Research
AUTHOR — AUTEUR
G. Siebert, A. Schmitt, R. v. Malorbie and E. Adloff
TITLE IN ENGLISFi — TITRE ANGLAIS
THERMAL DENATURATION OF ENZYME PROTEINS OF COLD-BLOODED ANIMALS
TITLE IN FOREIGN LANGUAGE — TITRE EN LANGUE Ec TRANGekRE
Thermische Denaturierung von KalthlUter-Enzymproteinen
REFERENCE — RFÉRENCE (NAME OF BOOK OR PUBLICATION — NOM DU LIVRE OU PUBLICATION)
1
Separatum EXPERIENTIA 16, 491 (1960)
PUBLISHER — IfOITLEUR
BirkhUuser Verlag
DATE
CITY — VILLE
Basel (Schweiz
REQUEST RECEIVED FROM
Mr. Paul Larose
Fisheries
DEPARTMENT
YOUR NUMBER
VOTRE DOSSIER NC)
REÇU LE
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L.
IC
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50.5-200-10.6
P. 491
OUR NUMBER
n
6657
Elisabeth
TRANSLATOR
Czeija
TRADUCTEUA
•
DATE RECEIVED
Vol.16, 1960
NOTRE DOSSIER N-
REQUIS PAR
MINISTàRE
PAGES
Date sent:
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31 August, 1964
DATE COMPLETED
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Reprint
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EXPERIENTIA 16, 491 (1960)
Birkhguser Verlag,
Publishing House:
Basel (Switzerland)
THERMAL DENATURATION OF ENZYME PROTEINS OF ODLD-BLOODED ANIMALS
The adenosine triphosphatase from carp musculature
1
shows a remarkably high heat-lability (PARTMANN ). Experiments for applying a heat-denaturation to the purification of cathepsin from cod musculature were unsuccessful, (SIEBERT and v. MALORTIE 2 ). Codfish, "Gadus callarias
(u morrhua) L.", usually lives at a water temperature of
4.H5to f 10 0 C. The question arises ) therefore, as to
whether or not it is a general characteristic of the
enzyme-proteins of the cold-blooded animals to become denaturized at considerably lower temperatures than this is
the case for enzymes of warm-blooded animals, in general
experience.
The experiments described below were under-
taken in order to clarify this question.
Methodology. .
In genera4thermal denaturations of
dissolved proteins are made in such a way that duration
of heating as well as the temperature reached are of importance for the effect.
In the tests to be described
here it proved useful to eliminate the time factor by
means of the principle of having the desired temperature
act for full 60 min. in a perfusion apparatus filled with
water.
Uontrol tests indicated that
in general 10 - 15
min., yet 30 min. at the most, are sufficient to produce
a certain extent of denaturation, which is not changed
further essentially by extending the heating period to
60 min. After heat influence as defined, the measurable
TABLE I. Activity and heat denaturation of various enzymes in cod musculature.
I1
1
I!
I
I
1
Activity
Measuring
Enzyme
Source of enzyme
Temperature
o,u
Specific
Total
uMol/h/mg
/uMol/h/g
/
Protein
Fresh weik'gt
2.9(tyrosine)
Temperature at which
50% denaturation
occurs after duration
of action of 1 hour
0.17
44
5.3
52
Cathepsin, raw extract
Cod muscle
37
Cathepsin, partly purified
Cod muscle
37
-
Glycyl-glycim-dipeptidase
Cod muscle
40
420
158
42
Cattle. muscle
40
185
35
46
II Lactic acid dehydrogenase
Cod muscle
25
20 100
750
52
' Aldolase
Cod muscle
25
1 250
43
i 1
.-F-
Triose phosphate dehydrogenase
1
I Encase
Cod muscle
25
4 6 00
180
51
Cod muscle
25
3 080
128
45
! Pyruvate kinase
Cod muscle
25
3 410
141
51
Cod muscle
25
202
1
i
I
i,
G]
, vcyl-glycine-di -rDeptidase
1
Ii isocitrico-dehydrogenase
8.4
30
1
IN)
-
enzyme activity w a s expressed in % of the initial value
and plotted graphically against the temperature used. The
temperature at which 50% of the initial activity of the
enzyme concerned are destroyed, which had been obtained
through extrapolation of at least 4 points of measurement,
lying on a straight line, served as the criterion for the
thermal resistanee.
Deep-frozen fillet of cod, freshly
caught (at sea), which was homogenized with 1% KC1-solution
for extracting the enzyme proteins of the glycolysis, and
with 1% LiBr-solution for extracting proteolytic enzymes ,.
was used as research material. Determining activity of
enzymes: Cathepsin according to LASKOWSKI 3 , glycy],-glycinedipeptidase according to SMITH 4 , ninhydrin reaction
according to MOORE and STEIN',•isicitrico-dehydrogenase
according to SIEBERT et al. 6 lactic acid dehydrogenase
according to BUCHER et al0 7 aldolase, triose phosphate
;
dehydrogenase and pyruvate kinase according to BUCHER et
al.
enolase according to BUCHER 9 , determination of protein according to LOWRY et al. 10 .
Results. The research contained in Table I show
that the range of 50% denaturation fluctuates very midely,
between 30 and 52 0 C. The adenosine triphosphatase
examined by PARTMANN falls into the lower range, with
aboui 38 ° • Hence, it is impossible to derive theoretical
principles from these data with regard to the mode of life
of cold-Uooded animais. It will only be possible to discuss protein-chemical consequences concerning the especially low thermal stability (as, let . us say, in contrast with
the highly heat-resistant enzyme proteins of thermophile
bacteria) when the enzymes concerned will be available in
purified form.
We have observed purifying powers due to
•t•'•
-4lactic acid dehydrogenase (2-fold),
.111
hePtinu
enolase (2.2-fold), pyruvate kinase (2.8-f o: ld), and
triose phosphate dehydrogenase (3.5-fold), but did not
follow this up any further. Among the different possibilities of explaing why purified cathepsin (SIEBERT
2
and v. MALO1TIE ) shows a greater heat resistance than
the raw extract present of the enzyme does, none is
supported by the experimental experiences existing thus
far.
As is to be expected, the thermo-dynamic qualities of the enzymes of cold-blooded animals are equal to
those of the enzymes of the warm-blooded animals.
This
TABLE II. Q 10 -values of two enzymes from cod musculature
Measurementrange in ° C
Glycylglycine-
Cathepsin
partly
used for the
calculation
dipeptidase
purified
from 37 to 27
1.66
2.03
1.86
32
22
27
17
1.97
22
12
2.13
17
7
2.17
12
2
2.67
7
- 3
2.13
3
2.41
-13
2.45
-
follows from Table II where the temperature coefficients
( .1 () are compiled for glycyl-glycine-dipeptidase and for
cathepsin from cod musculature. The value for cathepsin
1)24_
which is out of line in the measurement range 37 - 27 ° 0 )
as it shows relatively too little conversion at 37 0 C )
might be traced back to an already beginning thermal denaturation, although, according to experience,
in the
case of the enzymes examined here, the presence of substratum may provide protection, to a certain extent,
from heat denaturation.
It is pointed out with gratitude that these investigations were supported by a grant from the Bundes-
wirtschaftsministerium (federal department of industry),
Bonn. We thank Mr. Degener, who is with the Hanseati-
sche Hochseefischerei A.G. company, Bremerhaven-F.,
for having provided us with the research material.
G. SIEBERT,
A. SCHMITT,
R. v. MALORTIE and E. ADLOFF
Physiologisch-chemisches Institut der Universitilt Mainz (Physiological-chemical institute of the
University of Mainz (Germany), 14 July, 1960.
SUMARY
The temperature, which leads to 50% reduction
of catalytic activity by heat denaturation, has been
determined for 8 different enzymes from cod muscle, as
being in the range between 30 and 52 0 C.
Therefore-
there are no indications of a generally different heat
resistance of enzymes from cold-blooded animals as
compared with those from warm-blooded animals.
*
The
Translator's note: This Summary is in English in
the original text.
- 6
same conclusion is derived from calculations of Q10values, measured between -I- 37 and -- 13 0 C for
cathepsin and glycylglycine dipeptidase.
1 G. NEMITZ and W. PARTMANN, Z. Lebensm.-Unters. Forsch.
(J , food research), 109, 121 (1959).
2 G. SIEBERT and R. v. MALORTIE, unpublished.
3 M. LASKOWSKI, Methods Enzymol. 2, 27
(1955).
4 E. L. SMITH, Methods Enzymol. 2, 93 (1955).
5 S. MOORE and W. H. STEIN, J. biol. Chem. 211, 907 (1954).
6 G. SIEBERT, J. DUBUC, R. C. WARNER and G. W. E. PLAUT,
J. biol. Chem. 226, 965 (1957).
7 A. DELBRUCK, E. ZEBE and T. BUCHER, Biochem. Z. (Bio-
chemical J.) 331, 273 (1959).
8
R. SCHOLZ, H. SCHMITZ, T. BUCHER and J. O. LAMPEN,
Biochem. Z. (Biochemical J.), 331, 71 (1959).
9 T. BUCHER, Methods Enzymol. 1, 427 (1955).
10 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J.
RANDALL, J. biol. Chem. 193, 265 (1951).