IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 The Inhibitory Effect of Some Benzoic Acid Derivatives on Human Erythrocyte Catalase Activity Z.A.Ali Department of Chemistry , College of Education-Science ,Unive rsity of Salahddin-Hawler Abstract In order to study the kinetic of human erythrocytes catalase a well –known enzy me uses H2O2 as substrate as well as hy drogen accep tors, in non smokers and smoker individuals. Ant hranilic acid and p -Amino Benzoic Acid (PABA) were used to study their effect on the enzy me. The kinetic st udy confirmed that anthranilic is a non-comp etitive inhibitor with Km values of 0.95 and 1.0 for non smokers and smokers resp ectively (PABA) was found to be a comp etitive inhibitor with Vmax values of 8.0 and 8.9 for nonsmoker and smoker resp ectively Introduction he univalent reduction of oxy gen results in a series of cy totoxic oxy gen sp ecies. These highly reactive sp ecies can cause cell damage including lip id p eroxidation, inactivation of enzy mes, alteration of intra-cellular oxidation-reduction st ate and damage to DNA (1) (2). Catalase (human erythrocyte catalase E.C:1.11.1.6). Its functions include cataly zing the decomposition of hy drogen peroxide to water and oxy gen. ,also oxidizes different t oxins, such as formaldehy de, formic acid and alcohol. In doing so, it uses hy drogen p eroxide according to the following reaction (3): H2O2 + H2R 2H2O + R Catalase has one of the highest turnover rates for all enzy mes; one molecule of catalase can convert millions of molecules of hy drogen peroxide to water and oxy gen p er second (4). Catalase is atetramer of four p olyp eptide chains. It contains four p orp hy rin heme (iron) group s which allow t he enzy me to react with t he hydrogen peroxide (5) Ant hranilic acid (ortho amino benzoic acid) and its derivatives were used as a drug for p ain, temp erature (p y rexia) and inflammatory p rocesses, among non st eroidal anti inflammatory drug agents (6) ( 7). Para amino benzoic acid (PABA) is a co-enzy me associated with the vitamin B-comp lex forms p art of t he structure of folic acid and it thought as a vitamin within a vitamin. Para amino benzoic acid st imulates the intest inal bacteria enabling them to p roduce folic acid, which in turn aids in the p roduction of p antot henic acid .PABA, also help s to maintain healthy intest inal micro-flora. As a coenzy me PABA functions in the breakdown and utilization of p roteins and in the formation of red blood cells (8) (9). T IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 The objectives of the p resent st udy are to evaluate the lip id p eroxidation marker M alondialdehy de (M DA) in sera of non smokers and smoker's healthy individuals and to indicate the effect of anthranilic acid and Para amino benzoic acid on erythrocyte catalase activities, also to evaluate Km and Vmax in the p resence of the above comp ounds. Experimental part All reagents were of highly analytical grade. Blood samples were collected from healthy individuals; the erythrocytes were obtained by centrifugation of blood after coagulation at 2500rp m for 30 min at room temp erature. Hemoly sate of RBC s was obtained by washing the erythrocytes three times with normal saline solution (0.9%NaCl). Hemoly sate was p repared by adding four p arts volume of dist illed water to one sediment volume of erythrocyte. A chloroform-ethanol extract was p repared by adding (0.5ml) hemolysate to (3.5ml) ice cold DW , followed by (1ml) ethanol and (0.6ml) chloroform. Aft er centrifugation, 1:500 A dilution of this concentrated hemolysate was p repared with p hosp hate buffer immediately before the assay. De termination of serum Malondialdehyde (MDA) le vels The determination is based on the formation of colored comp lex up on the reaction of M DA with thiobarbituric acid (TBA) according to the method described by Rehnerona et al .The absorbance of complex was measured at 532 nm (10). De termination of e rythrocyte catalase activity The assay of catalase was p erformed on the ability of the enzy me to decompose H2O2 to give water and oxy gen. This assay was based on the reduction in the absorbance of hydrogen p eroxide at 240nm. The difference in absorp tion (∆A 240 ) p er unit time is a measure of catalase activity (11). -Prep aration of st ock solution of anthranilic acid and Para amino benzoic acid with concentration of 10 -2 mM of each:- Four different dilutions from organic comp ound were prepared by the following st eps: From the st ock (10 -2 mM ),one ml was transferred to 100ml volumetric flask and comp leted with ethanol to the mark to get 10 -3 mM concentration,also 10 -4 , 10 -5 and 10 -6 dilutions were prepared . Determination of percentage inhibition :- Using differ ent concentrations of anthranilic acid and Para amino benzoic acid , while the concentration of the substrate [S] was fixed to get t he percentage of inhibition or activation, accordin g to the equation: %Inhibition=100-(activity with inhibitor / activity without inhibitor) x 100 The inhibitor concentration that gives the highest p ercentage of inhibition was used throughout the study to obtain the ty p e of inhibition. De termination of type of i nhibition It was carried out by using different concentrations of substrate, while there are fixed concentrations of anthranilic acid and Para amino benzoic acid. The same method of catalase activity was used by utilizing the same concentrations of subst rates without using anthranilic acid and Para amino benzoic acid. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VOL.22 (3) 2009 The effect of ethanol, which was used as a diluent , was determined by adding a quantity equivalent t o the samp le and all st eps completed as in the method of determination of ery throcyte catalase activity (12). Re sults and Discussion Lip id p eroxidation marker M alondialdehy de (M DA) in healthy non-smokers were found to be 10.3 mM /dl ,while it was found to be 18.5mM /dl in healthy smoker individuals. The high levels of M DA in sera of smokers over non smoker could be due to the damage in the tissue caused by over generation of free radicals in the body of smoker compared to non smoker; also research has shown that many diseases are the direct results of free radicals on human body (13)(14) Figure (1) and (2) showed M ichaelis-M enten for erythrocyte catalase in non smokers and smokers resp ectively. The values for Vmax were (8 U/g) and (9.5 U/g) resp ectively. The Km values were 0.5 and 0.58 for non smokers and smokers resp ectively The catalase activity increased in erythrocyte of smoker comp ared to non smokers is due to the increase in free radical generation in smokers which was shown in the p resent st udy accomp anied with the increase in M DA concentration to counter balance by the increase in catalase activity esp ecially in erythrocytes which are rich in this enzy me (Chance . etal) (15) also Boon .etal rep orted that the elevated antioxidant enzy mes defence sy st ems in smokers erythroctes for p rotection against oxidative st ress (16) The effect of anthranilic acid on erythrocyte catalase activity of non smoker and smoker is shown in figure (3). Ant hranilic acid showed an inhibitory effect ( 34.2 ,40.9 ,62.7 ,60 %) and (41.6,40.3,37.1,53.7%) at concentrations (10 -3 ,10 -4 ,10 -5 and 10 -6 mM ) of non smoker and smoker resp ectively. The effect of Para amino benzoic acid on erythrocyte catalase activity of non smoker and smoker is shown in figure (4). Para amino benzoic acid showed an inhibitory effect (58.2, 40.3, 38.2, and 46.1%) and (65.9, 32.8, 44.4, 39.4 %) at concentrations (10 -3 , 10 -4 , 10 -5 and 10 -6 mM ) of non smoker and smoker resp ectively. The effect of the solvent ethanol showed a negligible inhibitory effect on erythrocyte catalase activity . The percentage of original activity is considered to be 100%. Figure (5) and (6) showed that Ant hranelic acid is a non-comp atative inhibitor for the erythrocytes catalase of non smokers and smokers resp ectively according to linweaver-barke p lot with Km values of 0.95 and 1.01 for non smokers and smokers resp ectively. From figure (5), the Vmax for the uninhibited enzy me is 9.1 and 3.9 for the inhibited enzy me in figure (6), the Vmax was found t o be 8.0 for smokers and 2.5 for the inhibited enzy mes in smokers. Figures (7) and (8) showed the linweaver – burk p lot for nonsmokers and smokers resp ectively which indicate a comp atative inhibition of PABA on catalase activity with the same Vmax (8.0 for both the inhibited and non inhibited catalase in non smokers) the Km values for the uninhibited is 1.0- while for the inhibited enzy me is 2.5 in non smokers from figure (8) the V max is 8.9 for the erythrocyte smokers with Km values of 1.0 for the uninhibited enzy me and 2088 for the inhibited enzy me. The complete M echanism of catalase is not currently known, t he reaction is believed to occur in two st ages: H2O2 + Fe (III)-E _____ H2O +O=Fe (IV)-E H2O2 + O=Fe (IV)-E ____ H2O + Fe (III)-E + O2 IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 (Where O=Fe (IV)-E rep resents the iron center of the heme group att ached to t he enzy me (17). A st udy conducted on the inhibitory effect of azide could be due to the removal of some catalase-Fe (III) from the reaction medium as the catalase – Fe (III)-azide complex (18). Re ferences 1 -Prashant A.V., Harishchandra H.,Dsouz a V.and Dsouz a B.(2007):Indian Journal of Clinical Biochemistry ;22(1):131-134. 2 -Chence B.,Sies Z.H.and Boveris A.(1979):Phy soil.Rev.;59:527-605. 3 -Berg J.M .,Ty moczko J.L.,Stry er L.(2002):Biochemistry ,Fifth Edition , WH Freeman and comp any ,New York and London:506,614. 4 -Hugo A. (1974): M ethods of enzy matic Analysis; 2:673-684. 5 -Steritwieser A.and Heathcock C.h.(1976):Introduction to organic chemistry ,M acmillan publishing Co.,In.C.New York and London:966. 6 -Fadeyi O.O.,Obafemi C.A. ,Adewunmi C.O.and Iwalewa E.O.(2004):African Journal of Biotechnology ;3(8):426-431. 7-Garrett R.H.and Grisham C.M .(2005):Biochemist ry ,Third Edition ,BRooks/Cole;Thomson;556-575. 8 -Akberova S.I.(2002):Biology Bullst in,29(4): 9 -Voet D.and Voet J.G. (2004): Biochemistry ; Third Edition,John Wiley and Sons,InC:768. 10 -Acbi H.(1974) :Practical Hematology ,Fifth Edition Edinburg , Churchill-Livingst one 11 -Reitman S.and Frankel S.(1957):Amer J Clin Path;92:60-67. 12 -Esterbauer H.,Gebicki J.,Puhl H. and Yungens G.(1992);13:341-390 13 -Flagg E.W.,Coates R.J. and Greeberg R.S.(1995): Journal of the American College of Nut rition; 14:419-426. 14 -Lardinois O.M .,M estdagh M .M .and Rouxhet P.G.(1996) :Biochem. Biophy s. Acta; 1295:222-238. 15 -chance B., Sies H., and Boveris , A.(1979): Hy drop eroxide metabolism in mammalian organs ., p hy sio .Rev., 59:527-605. 16 -Boon E.M .,Downs A. and M arcey D.(2007):”Catalase Structure Tutorial Text “. 17 -Lardinois O.M . and Rouxhet P.G.(1996):Biochem.Biophy s.Acta;1298:180-190. 18 -Aksoy Y.,Balk M .,Ogus H. and Ozer N.(2004):Turk J.Biol;28:65-70. 2009) 3( 22مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد الكاتلیز في انزیم فعالیة فيثیر التثبیطي لبعض مشتقات حامض البنزوك أالت كریات الدم الحمراء البشریه زیان عبد اللة علي هولیرجامعة صالح الدین ،علوم /ةتربیال ةكلی ،قسم الكیمیاء الخالصة للهیـدروجین مادة اساس وكـذلك مسـتقبال H2O2 المعالجل دراسة حركیة انزیم كتلیز كریات حمراء الدم البشري بأ ست باسراف مدخنین والمدخنینالفي االفراد غیر .هذا االنزیم في ات لدراسة تاثیرهملمعامینو حامض البنزوك است–حامض االنثرانیلك و بارا لغیــر المــدخنین والمــدخنین علــى 1.0و Km 0.95تنافســي بقــیم ل ال اكــدت الدراســة الحركیــة ان االنثرانلــك مثــبط لغیـر المـدخنین والمـدخنین 8.9و Vmax 8.0 مـع قـیم ل اتنافسـی االتوالي ، بارا امینو حامض البنزوك وجد لكي یكـون مثبطـ على التوالي IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 Fig.(7): Line weaver-Burk plots for Para amino benzoic acid on e rythrocyte catalase activity of non smoker. Fig.(8): Line weaver-Burk plots for Para amino benzoic acid on e rythrocyte catalase activity of smoker. 0 0.2 0.4 0.6 0.8 1 -4 -3 -2 -1 0 1 2 3 4 1/[S] 1 /V m a x with out inhibitor with inhibi tor 0 0.2 0.4 0.6 0.8 1 -4 -3 -2 -1 0 1 2 3 4 1/[S] 1 /[ V m a x ] with out inhibitor with inhibi tor