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AAbleves ". •': (-) ••• • • c. (,,..... 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 :,,.. /' /1•'i':$ 4/(., ' • . • I . 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 5 [-I L. IC s-- 1A 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: 769-18-14 31 August, 1964 DATE COMPLETED REMPLIE LE 5/111/1965 /:• • Reprint ,.(9/3 /1/0. 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).