1) When guinea pig serum was warmed at about 65°C,
part of its protein became denatured resulting in the formation of a colloidal component C, which has approximately the same electrophoretic mobility as that of &#945;-globulin. 2) The electrophoretic examination shows that the serum heated at 65°C after adding the glucose is devoid of colloidal component C. 3) The inhibitory action of sugar and polyatomic alcohol on the heat inactivation of complement is galactose&#62; saccharose &#62; glucose&#62; sorbit &#62; mannit &#62; glycocoll&#62; glycerin in descending order.
The inhibitory action of sugar and poly atomic alcohol on
molecular aggregation by heating is of the same order as complement activity.</p

Mochizuki, Yoshio

Ogata, Masana

Okayama University Digital Information Repository

Studies on the Inhibitory Action of Sugar and Polyatomic Alcohol for the Heat-inactivation of Com­plement

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 10, Issue 2 1956 Article 4APRIL 1956Studies on the Inhibitory Action of Sugar andPolyatomic Alcohol for the Heat-inactivationof Com-plementMasana Ogata∗ Yoshio Mochizuki†∗Okayama University,†Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on the Inhibitory Action of Sugar andPolyatomic Alcohol for the Heat-inactivationof Com-plement∗Masana Ogata and Yoshio MochizukiAbstract1) When guinea pig serum was warmed at about 65◦C, part of its protein became denaturedresulting in the formation of a colloidal component C, which has approximately the same elec-trophoretic mobility as that of α-globulin. 2) The electrophoretic examination shows that theserum heated at 65◦C after adding the glucose is devoid of colloidal component C. 3) The in-hibitory action of sugar and polyatomic alcohol on the heat inactivation of complement is galactose>saccharose> glucose> sorbit>mannit> glycocoll> glycerin in descending order. The inhibitoryaction of sugar and poly atomic alcohol on molecular aggregation by heating is of the same orderas complement activity.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLSTUDIES ON THE INHIBITORY ACTION OF SUGARAND POLYATOMIC ALCOHOL FOR THE HEAT-INACTIVATION OF COMPLEMENTByMasana Ogata and Yoshio MochizukiDepartment of Public Health and Hygiene, OkayamaUniversity Medical School(Director: Prof. Dr. M.Ogata, and Prof. Dr. K.Ohtahara)Received for publication on January 31 1956.IntroductionWhen complement is warmed to about 56 0 C, it becomesopalescent. If the heating is prolonged or the temperature israised still higher, the cloudiness and viscosity increase andthe entire sample may become gel.Van der Scheer showed)) that the horse serum, whenwarmed to about 65 0 C, becomes denatured to form a colloidalcomponent C, having approximately the same electrophoreticmobility as that of the p-globulin.It was published that the sugar and polyatomic alcoholhad an inhibitory action for the heat denaturation of biologicalactive substances, i. e., diphtheria toxin2), antitoxintl ), enzyme-l),hemagglutinin ii)()7) and complementRHll •L. Silber fl ) found in 1932 that thermostability of comple-ment was increased by sugar. This discovery was confirmedand extended by M. Ogata, T. Ohkawa, M. Ogata10) and M. Ratanall).The purpose of the present study is to show the effect of sugarand polyatomic alcohol on the formation of a colloidal compo-nent C, by means of electrophoretic analysis and measurementof turbidity.Materials and MethodsThe complement used in this experiment was that containedin fresh serum taken from guinea- pigs.Complementary activity was measured by the initial hemo-lysis method advocated by Ecker, Pillimer, Wertheimer andGradisl2). Each sugar and amino acid, was tested for its inhibi-1Ogata and Mochizuki: Studies on the Inhibitory Action of Sugar and Polyatomic AlcoholProduced by The Berkeley Electronic Press, 1956Studies on the Inhibitory Action of Sugar etc. 83tory action on heat denaturation by the following method.The calculated weight of sugar and amino acid was addedto 0.5 ml complement, then the mixture was shaken and incu-bated at respective temperature (54°C, 56°C, 58°C, 60°C, 62°C,64 °C, 65°C) for 15 minutes in a water bath.Electrophoretic examination;Electrophoretic examination has been made with the "Hi-tachi A type" electrophoretic apparatus. Photographic recordswere taken by Svensson's methodl3). All observations have beenmade on the migration of protein in the buffer. Before electro-phoresis, the samples of guinea-pig serum were diluted withequal volume of a buffer solutionand then thoroughly dialyzedagainst large volume of buffer. The ionic strength of this bufferwas 0.2 with respect to phosphates; its pH was 7.7. Afterthe first dialysis of 48 hours, the electrophoretic examinationhas been made.Turbidity analys1~s ;The strength of light scattering has been measured withnephro-colorimeter.Experimental resultsA) The electrophoretic examination;The electrophoretic patterns obtained from the heat-inacti-vated guinea-pig serum and that after being added glucose,were shown in Table I and Fig. 1. In Fig. I (B) and (D) the moststriking feature is a component C, that dominates the patternsof sera heated at 65°C. The C is a colloidal aggregationproductresulting from denaturation of guinea-pig serum (complement).The C peak moves in the electric field at practically the samerate as the a-peak appearing in native serum. The electro-phoretic examination shows that the serum heated at65°C after being added the glucose is devoid of Ccomponent.(Fig. 1 E)(cf.)In order to remove the sugar from the serum, the serumwas precipitated by saturation with natrium sulfate, and wasmeasured by electrophoretic method. From Fig. 1 (A), (C) and2Acta Medica Okayama, Vol. 10 [1956], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss2/484 M.Ogata and Y. Mochizuki:Fig. 1 (B)1 (D), the percentage. of serum components (albumin,a-, f3-, and r-globulin) measured by electrophoresis were appro-ximately the same value as that of serum components precipi-tated by natrium sulfate.Table 1. The percentage composition of thecomponents of guinea-pig serum.Percentage of components.Albuminl a-glob.orC i f3-g lob./ ')'-glob.Native complement(A) (Normal guinea-pig serum)(B) Complement heated for 15 minutesat 65°C.(C) Complement precipitated by satu-ration with natrium sulfate.(D) Complement heated for 15 minutesat 65°C and precipitated by satu-ration with natrium sulfate.(E) Complement when added glucoseheated for 15 minutes at 65°C andprecipitated by saturation with nat-rium sulfate.61.523.064.625.059.613.873.012.371.415.813.110.811.411.64.012.33.613.2Phosphate buffer, pH 7.7, Ionic strength 0.2,From the above results it was inferred that glucose inhibi-ted the molecular aggregation of serum protein and the forma-tion of colloidal component C. These phenomena were also ob-served by turbidity measurment.B) The turbidity measurement;Five portions of complement heated to various tempera-tures (54°C, 56°C, 58°C, 60°C, 62°C) for 15 minutes, weretested for their activity and turbidity.Turbidity was expressed in terms of relative turbidity, i. e.,turbidity reading of heat inactivated serum.relative turbidity turbidity reading of native serum.Calculated weight of each sugar and amino acid was addedto complement, heated at respective temperature (54° C, 56° C,58°C, 60°C, 62 C C), and the turbidity was measured with ne-phro-colorimeter.The results were shown in Table 2 and Fig. 2. Column V -3Ogata and Mochizuki: Studies on the Inhibitory Action of Sugar and Polyatomic AlcoholProduced by The Berkeley Electronic Press, 1956Studies on the Inhibitol'Y Action of Sugar etc.Fig. 1. Electrophoretic patterns of guinea-pig serum.85A) The pattern of native complement.(guinea pig serum)cB) The pattern of complement afterbeing heated for 15 minutes at 65°C.EAIIt~ C) The pattern of complement precipi-tated by saturation with natriumsulfate.f3 'If £~cD) The pattern of complement afterbeing heated for 15 minutes at 65°Cand then precipitated by saturationwith natrium sulfate.f..All.f\ E) The pattern of complement whenglucose added after being heated for15 minutes at 65°C and then prec-(3 ~ ipitated by saturation with natriumsulfate.X, the last series (VIII) in table 2 shows the relative turbidityof complement heated at various temperature. Column V - X,series I - VII shows that of complement after adding the calcu-lated amount of sugar and poly atomic alcohol so as to be 10times as much as the serum osmotic pressure. Column IV showsthe maximum temperature allowed for the complement activity.The Fig. 2 shows the results of Table 2. Heating temperatures4Acta Medica Okayama, Vol. 10 [1956], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss2/4Complement 11 Gram IMOC 56°C 58°C 60°C 62°C 64:°C15 min 15 min 15 min 15 min 15 min 15 minMaximumMinimum inhibitory+ added Minimum hemolytic tempera- Relative Relative Relative Relative Relative RelativeSugar, : t 0 5 I hit' unit after ture forPit . I 0 . memo Y IC heating inactiva-o ya omlC . turbidity turbidity turbidity turbidity turbidity turbidityalcohol and Iof comple- unit (ml) 56°C for tion ofAmino acid \ t 15 mint. comple-I men mentComplement I+ 0.3 0.03 0.03 63°C 1.0 1.0 1.0 1.0 1.1* 1.1*GalactoseComplement+ 0.45 0.04 0.04 62°C 1.0 1.0 1.0 1.1 1.2* 1.3SaccharoseComplement+ 0.3 0.03 0.03 62°C 1.0 1.0I1.0 1.1 1.1* 1.3GlucoseComplement+ 0.3 0.03 0.03 61°C 1.0 1.0 1.1 1.2* 1.3* 1.7SorbitComplement+ 0.3 0.03 0.04 59°C 1.1 1.1 1.2* 1.2* 1.7 1.7MannitComplement+ 0.1 0.03 0.03 57°C 1.1 1.2* 1.25* 1.35 1.7 2.0GlycocollComplement+ o.15(ml) 0.03 - 54°C 1.1* 1.2 1.35 1.4 1.8 2.1GlycerinNative- 0.03 -I55°C 1.1 I 1.2* 2.1 2.8 4.3 5.0complementTable 2. The relative turbidity of complement and complement heated at various temperatureafter adding sugar and pelyatomic alcohol.00~~o~~<+~~t:lP,~~o~c:N~e:* shows the maximum inhihitory temperature for inactivation of complement.5Ogata and Mochizuki: Studies on the Inhibitory Action of Sugar and Polyatomic AlcoholProduced by The Berkeley Electronic Press, 1956Studies on the Inhibitory Action of Sugar etc. 87Compl. +GlycerinCompl. +GlycocollCompl.+MannitCompl. +SorbitCompl. +SaccharoseCompl. +Glucose62505856Compl. +----....-::~--__~ ..::::. ____J Galactose64°.541.11.01.32.0Fig. 2. The nelative turbidity of complement and complement heatedvarious temperature after adding sugar and polyatomic alcohol.5.0 ------------ Nativle3 0 ~ comp ement. (Compl.)Vertical axis shows the relative turbidity. Horizontal axisshows the heating temperature.were shown on horizontal axis, and relative turbidity shownon vertical axis. The increase of turbidity caused by heat inac-tivation has not been observed in the serum heated after addingthe sugar and poly atomic alcohol. These results correspond tothe colloidal C peak formed by heat inactivation by electro-phoretic method. The inhibitory action of sugar and polyatomicalcohol for molecu lar aggregation by heating is galactose>saccharose> glucose> sorbit > mannit> glycocoll> glycerinin descending order and same order as inhibitory action of heat6Acta Medica Okayama, Vol. 10 [1956], Iss. 2, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol10/iss2/488 M.Ogata and Y. Mochizukiinactivation of complement activity..The experiments show thermostability of complement in-creases by inhibitory action of sugar and polyatomic alcohol onmolecular aggregation by heating.Summary1) When guinea pig serum was warmed at about 65 0 C,part of its protein became denatured resulting in the formationof a colloidal component C, which has approximately the sameelectrophoretic mobility as that of a-globulin.2) The electrophoretic examination shows that the serumheated at 65°C after adding the glucose is devoid of colloidalcomponent C.3) The inhibitory action of sugar and polyatomic alcoholon the heat inactivation of complement is galactose> saccharose> glucose> sorbit > mannit > glycocoll> glycerin in descend-ing order.The inhibitory action of sugar and poly atomic alcohol onmolecular aggregation by heating is of the same order as com-plement activity.The authors wish to express our thanks to Prof. M. Ogata, forand Prof.K. Ohtahara for suggesting this investigation as well as for constant guidancein the course of the work.References) Van der Scheer, J., Wycko!!, R. W. G. and Clarke, F. L.: J. ImmulloI. 40,39, 1941. - 2) Kulikow, W. u. Smirnow: Zeitschr. f. d. expo BioI. U. Med. 4,14, 83, 1927. - ~) Kulikow, W. U. Sekretewa: Compt. Soc. BioI. 97, 1339, 1927.- 4) Kulikow, W. U. Bobkowa: Zeitschr. f. d. expo BioI. U. Med. 4, 25, 197.- [» Silber, L. U. Nikolskaja: Zeitschr. f. d. Immu. Forsch. 59, 66, 1924.-6) Silber, L.: Zeitschr. f. d. Immu. Forsch. 63, 506, 1928. - 7) Silber, L.:Zeitschr. f. d. Immu. Forsch. 64, 167, 1929. - 8) Silber, L.: Zeitschr. f. d.Immu. Forsch. 65, 285, 1930. - 9) Silber, L.: Zeitschr. f. d. Immu. Forsch.67, 514, 1932. - lO) Ogata, M, Okawa, T. and Ogata, M. : Okayama-Igakkai-Zasshi. 59, 654, 1947. - l1) Hatano, M.: J. Hiroshima Med. Ass. 6, 11, 53,65, 1953. - J. Hiroshima Med. Ass. 7, 5, 36, 43, 1954. - )2) Ecker, E. E.,Pillemer, L., Wertheimer, D. and Gradis, H.: J. ImmunoI. 34, 19, 1938. - ];J)Svens-son, V. H.: Kolloid Zeitschrift. 90, 2. 141. 1940.7Ogata and Mochizuki: Studies on the Inhibitory Action of Sugar and Polyatomic AlcoholProduced by The Berkeley Electronic Press, 1956

Studies on the Inhibitory Action of Sugar and Polyatomic Alcohol for the Heat-inactivation of Complement

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Studies on the Inhibitory Action of Sugar and Polyatomic Alcohol for the Heat-inactivation of Com­plement

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Studies on the Inhibitory Action of Sugar and Polyatomic Alcohol for the Heat-inactivation of Complement