IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 The Study of Serum GOT(Glutamic Oxalacetic Transaminase) Activity and Some kinetic Parameters in Patient with Pulmonary Tuberculosis P. A.Ismahil , L. M.Hassan Department of Chemistry ,College of Education Science,University of Salahaddin Abstract In the present study, serum GOT(Glutamic Oxalacetic Transaminase) was purified, the purified enzyme showed the maximum activity at 37ºC and pH 7.5. During purification of serum GOT ion exchange chromatography lead to four separate forms (termed I, II, III and IV). GOT II with the highest specific activity was pure after chromatography on Sephacryl S- 300. . S.GOT levels were investigated in serum samples from patient with pulmonary tuberculosis .The S.GOT levels were determined at and 37ć .The study revealed that the serum GOT activity was higher in patient of tuberculous pulmonary than in control subjects.The mean serum GOT activity in the patients group was(90±8 IU/L) as compared to control group (27±0.65IU/L) showing a highly significant (p<0.001)difference .The study was concentrated to comprehensive measurement of the rate reaction kinetics(Km,Vmax,Hill coefficient),the first order forward rate constant k1 and half life t1/2 of the enzyme reaction in both normal and tuberculosis serum to evaluate any change In the number and structure of the active site then the rate of enzyme –substrate binding. Key words :Glutamic Oxalacetic Transaminase.Tuberculosis Introduction Tuberculosis is a chronic bacterial infection caused by Mycobacterium tuberculosis and characterized by the formation of granulomas in infected tissue and by cell –mediated hypersensitivity . The usual site of diseases is the lung ,but other organs may be involved [1,2,3],Tuberculosis has emerged as one of the most lethal diseases man has sever faced in short span of time become a major health problem in the third word. The severity of the diseases can be judged by the fact that it affects all ages [4,5,6].Mycobacterium tuberculosis is responsible for more morbidity in humans than any other bacterial diseases.M ycobacterium infects 1.1 billion people per year which is equal to 33% of the entire word population [7,8,9] Glutamic oxalacetic transaminase is a tissue enzyme that catalyzes the transfer of amino and keto group between alpha-amino acid and alpha-keto acid .The GOT exist in the tissues of many organs .Necrotic activity in these organs causes a release of abnormal quantities of enzyme into the blood .An increase in serum GOT activity has been described in several diseases as myocardial infarction. GOT activity is also increased with various types of liver diseases like viral hepatitis or in liver damage and some time with renal diseases .AST or necrosis and inflammation of heart,liver and muscle cells [10,11,12,13] Experimental *Samples collection and Separation Blood analysis is usually done on venous of capillary blood.60 samples of the blood were through out the investigation of GOT enzyme activity ,35 of these samples were of IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 tuberculosis ,while 25 of these samples were of healthy individuals .5 ml of blood has been collected and allowed standing at room temperature until it has clotted. Restriction of the clot may be assisted by gentle loosing it from the walls of the container. The serum was separated by centrifugation at 3000 r.p.m for 15 minutes.Separated serum was used on the same day of determination Chemicals All chemicals used were of high analysis grade and parched from BDH ,FLUKA *Instruments and Apparatus -Sepctrophotometer, supertonic 601 -Philips pH-meter -Thermostatic water bath -Centrifuge T.5 *Methods A-Estimation of S.GOT activity S .GOT activity was determined by measurement of oxaloacetate produced during the assay according to the method of Bergmeyer and Bernt (1965). The samples were incubated at 37◦C for 1 h in tubes containing 100 μM aspartic acid, 2μM α-ketoglutaric acid, 20mM sodium phosphate buffer, pH 7.5 and appropriate amount of enzyme to give a final volume of 1.0ml. The reaction was stopped by the addition of 1ml 2,4-dinitrophenylhydrazine reagent and allowed to incubate 20min at room temperature and 10ml 0.4N sodium hydroxide was added. The absorbance was recorded at 505 nm. The enzymatically liberated oxaloacetate was calculated from a standard curve absorbed at 510 nm ,using series concentration of substrate[S] Fig(3). Purification of aspartate transaminase Step 1: Freshly drawn, heparinized human blood is centrifuged at 5000 rpm for 30 min, and the plasma is treated with ammonium sulfate. The protein fraction precipitating between 30 and 80% saturation is collected Step 2: The pooled supernatants (630ml) were placed in a 60°C water bath for 40min with frequent mixing and then centrifuged at 100OOg for 30min. Step 3: The supernatant from stage 2 was further purified by (NH4)2SO4 precipitation. Optimum precipitation occurred between 64 and 72% saturation, with an overall purification factor of 69 and a yield of 37%. and a yield of 30% were obtained. Protein precipitating overnight in the optimum range was collected and redissolved in 0.01 M-sodium phosphate buffer, pH7.4, dialyzed against the same buffer Step 4:DEAE-cellulose chromatography The dialyzed supernatant from Step 3 was applied directly to a DEAEcellulose column (20×2.6 cm i.d.) equilibrated with 50mM sodium phosphate buffer, pH 7.5. The adsorbed material was eluted with a stepwise gradient ranging from 0 to 0.4M NaCl prepared in the same buffer at a flow rate of 60ml/h and 10ml fractions were collected. Protein fractions exhibitingAST activity were eluted with 0.0, 0.05, 0.1 and 0.2M NaCl, respectively and designated AST I, II, III and IV, according to elution order. Step 5: Sephacryl chromatography AST II was applied to a Sephacryl S-300 column (95×1.6 cm i.d.) equilibrated with 50mM sodium phosphate buffer, pH 7.5 and developed at a flow rate of 60ml/h and 3ml fractions were collected. The AST II was eluted withthe same buffer. Protein determination Protein was determined either by measuring the absorbance at 280 nm (Warburg and Christian, 1942) or by the method of Bradford (1976) using bovine serum albumin as a standard IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 B-Estimation of physical constant (Km& Vmax) values The values of Km &Vmax was determined in normal and tuberculosis cases the same experimental protocol of activity was applied and the only difference is the use of different substrate concentration .The data obtained were examined for Km & Vmax by using different methods of p lotting data .Theses were of 1-Michaeles-M entin plot in which the rate of reaction(V)is plotted Vs [S],directly 2-Lineweaver-Burk plot a reciprocal plot of 1/V Vs 1/[S] has been applied C-Estimation of the rate association constant (K1),half-life time (t1/2) Following time course reaction ,the order of the enzymatic reaction (K1) and half life times(t1/2) were determined .The same protocol of the activity was applied at constant [S] but the time of incubation was varied (fig5,6) D-Estimation of the activation Energies(Ea*) The same protocol of GOT determination was adopted and the only difference is the use of various incubation temperature (fig7) Results and Discussion The purification of AST is summarized in Table 1. From the elution profile of the chromatography on DEAE-cellulose (Figure 1a), it can be seen that AST activity was detected in four peaks: the negative adsorbed fractions and the fractions eluted with 0.05, 0.1 and 0.2M sodium chloride and designated as GOT I, II, III and IV, respectively. Further purification was restricted to GOT II. A Sephacryl S-300 column (Figure1b) was used to obtain GOT II. with the highest possible specific activity (3.63 units/mg protein). The activity of GOT in tuberculosis was estimated quantitatively and compared with that of normal using developed calorimetric method .Data obtained revealed specific elevation of GOT activity (90±8.1 IU/L) in case of tuberculosis fig(2) comparing with normal individuals (27±0.65 IU/L) .This is due to that serum GOT level positively corrected to the diseases extent in tuberculosis of immunocomptenl patients .The presence of elevated enzyme activity in the plasma may indicate tissue damage accompanied by increased release of intracellular enzymes. Many diseases that cause tissue damage result in an increased release of intracellular enzyme in to the plasma .The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Thus ,the degree of elevation of a particular enzyme activity in plasma is often useful in evaluating the prognosis of the patient [15].The Km values for the serum GOT decreased in tuberculosis patient( 0.64 ±0.5mM)fig.4 table2) as compared to normal group(1.03 ±0.8mM) .This means that the affinity of the enzyme for their substrate was affected by the diseases and chemical structure (ionic state ) of the active sites become more suitable for the substrate for binding [16].While the Vmax for the serum GOT increased in tuberculosis patients (90±8 IU/L) (IU/L) (fig 3 table2) as compared to normal(27±0.65 IU/L) these results are acceptable because there is an increase in lymphocytes and macrophages activities and concentration in tuberculosis patient which affect the rate of general metabolism [17].So increasing GOT activities and concentration (number of active site).(Fig 5 table 3 )indicates that the GOT enzyme reaction in both normal and tuberculosis patients reaction are of the pseudo first order type from these figures, the forward reaction rate constant(association rate constant)(K1) and half time (t1/2) were estimated in both cases using the following equation The results showed that the (K1) for serum GOT increased in tuberculosis patient but (t1/2) decreased as compared with normal group(fig 6 table3), so one can conclude that the diseases may affect equilibrium constant(Keq) of (Ea*)complex formation through out increasing the association constant [18,19]. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI VO L. 23 (3) 2010 The results also showed that the tuberculosis affected (decreased) the half life time of serum GOT enzyme reaction . This may be due to the acceleration of their activities by increasing the GOT concentration and the affinity of the GOT to their substrate .The hill coefficient(n) value of the serum enzyme binding site to their substrate was estimated from hill plot fig6.The results (table3) revealed that there were no cooperation and no significant change in (n)value in the case of tuberculosis comparing with that of normal groups.( Fig7 table4) represent the Arrhenius plot for the serum GOT enzyme in normal and tuberculosis patient from the figures the energy of activation of the enzyme substrate reaction were estimated (table4).The results show that (Ea*)for enzyme reaction decreases in tuberculosis case .These results indicate that the diseases may affect the mechanism of the enzyme reactions, this predicts that there is another pathway for the enzyme-substrate complex((Ea*)[20] IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SCI VO L. 23 (3) 2010 References 1-Braunwald,B ; Martin ,M and Kasper,D.(1998).Harrisons principles of internal Medicine.2: 14 ed..MC.applet inlang.USA 2-Meena,V ; Sanjeev;Ashsh,M andAkshra,V(2004) "study of adenosine deaminase activity in pulmonary tuerculosis and other common respiratory diseases ".Ind. J. of clinical biochemistry 193(1) 129-131 3-Antonisks,D; Amin ,k and Barnes, P(1990) "pleuritis as a manifestation of reactivation tuberulosis.Am.J.Med. 89:447-450 4-Chan, C;Arnold Mand Mak, T(1987)"clinical and pathological features of tuberculous pleural effusion and its long term consequences." Respiration 91:106-109 5-Dunla, N Pand Brile ,DE(1992)."Immunology of tuberculosis .Med.Clin.North Am;77:1235-51 6-Sudre, P;Dam, GT and Koch ,A(1992)"Tuberculosis a global overview of the situation today .Bull WHO;70:149-159 7-Ellioh, A;Luo, N and Tembo, G(1990) "Impact of study B M J;31.412-415 8-Modilesky ,T;Sattler, F and Barnes, P(1991)"Myobacterial disease in patient with human immunodeficiency virus infection"Arch Inter. Med ,149:2201- 2205 9-Jonathen,E.G(2001)"Transmission of Myobacterium tuberculosis through casual contact with infectious.Archives of internal Medicine .161(8):2254-63 10-Iodar, K;(2002)."Tuberculosis Scientific Report.University of Wisconsin 11-Banks,B and Vernon ,C(1961)"Transaminating part 1 .The isolation of apoenzyme of glutamic-aspartic transamminase from pig heartmuscl.J.Chen. Socc.1698-1705 12-Dumitrn, I. ;Iordahescu, D. and Niculesu ,S.(1961)"L-Alanine:2- oxoglutaratetransfesrase chromatographi purification and crystallization of the enzyme 44-:317from seed of glycine hisp ida var cheepear"Rev.Roum.Biochem. 13-Tonhazy ,N. ;White, N. and Umbreite, W.(1960) "a rapid method for the estimation of the glutamic-aspartic transaminase in tissue and its application of radation." Arch. 42-6:.328Biohem. 14- Bergmeyer, H.U. and Bernt, E.(1965). Glutamate oxaloacetate transaminase. In: Method of enzymatic analysis, H.U. Bergmeyer (ed.), pp. 837–853. Acad press, New York. 15-Chmpe,P.C.and Harrvey,R.A. (1994)"Lippincotts illustrated ".Reviews Biochemistry .2 nd ed.lipinott.J.B. USA IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI VOL. 23 (3) 2010 ed th,52Varly,H.;Alan,H. and Maurice,B.(1980)."practical clinical chemistry ,Vol -16 .Willam Heinmann Medical Books LTD .London 17-Gyton,A.C.(1991) "Text book of medical physiology 8 th ed .W.B.Saunders.USA 18-Ross,E. ;Maguire,M. and Stugll,T.(1977)."Relationship between the β-adrenergi 75-:576252receptor and adenylate cyclase.J.Biol.Chem. 7407-:7736254Waelboeck,M.;Vanobbergh,E. and Demeyte ,D.(1979) J.Biol.Chem. -19 1789-:177366Nemthy ,G. and Cheraga, T.(1972) J.Phys.Chem -20 IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (1): Purification scheme for serum GOT II Recovery% Fold purification Specific activity(units/ mg) protein Total protein mg Total units Purification step 7.8 3.3 0.4 10 0.4 DEAE-cellulose 0.01 M NaCl (I) 32.1 5.25 0.63 26 16.4 0.05 M NaCl (II) 22.5 2.1 0.25 46 11.5 0.1 M NaCl (III) 25.5 1.44 0.173 75 13 0.2 M NaCl (IV) 27 30.25 3.63 3.8 13.8 Sephacryl S-300 AST II ∗One unit of GOT activity was defined as the amount of enzyme producing 1μmol oxaloacetate (pyruvate) per h under standard assay conditions. Table(2):Km and Vmax value of Normal& Tuberculosis S GOT Normal Tuberculosis Enzyme Vmax(IU/L) Km(mM) Vmax(IU/L) Km(mM) GOT 27 1.03 90 0.64 Table(3):Enzyme reaction parameters of Normal and Tuberculosis SGOT Hil Coefficient (n) t1/2 K1(min-¹) Enzyme Tuberculosis Normal Tuberculosis Normal Tuberculosis Normal 1.9 7 1.09 15.4 24.75 0.045 0.028 ِ◌GOT IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table(4):Activation energy of Normal and Tuberculosis S GOT Activation energy Ea Enzyme Tuberculosis Normal 7925.18 8882.18 GOT Fig. (1) (a): A typical elution profile for the chromatography of. S erum GOT on DEAE- cellulose column (20×2.6 cm i.d.) previously equilibrated with 20mM sodium phosphate buffer, pH 7.5 at a flow rate of 60 ml/h and 10ml fractions. (b) A typical elution profile for the chromatography of Serum GOT II DEAE-cellulose fraction on S ephacryl S-300 column (95×1.6 cm i.d.) previously equilibrated with 20mM sodium phosphate buffer, pH 7.5 at a flow rate of 60ml/h and 3 ml fractions. Absorbance at 280 nm (•—•)GOT ,activity IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig(2):Serum GOT leve ls in norm al and tuberculosis group 0 20 40 60 80 100 1 2 S .G O T a c ti vi ty ( IU \L ) Norm al Tuber culosis 27 90 Fig(3):Michae lis-M ente n plot for both normal and tuberculosis 0 20 40 60 80 100 120 0 1 2 3 4 5 6 [S]mM V I U /L Tubercul osis Normal Fig(4): Linwe ave r-Burk plot S.GOT for both tuberculosis and normal serum -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 -4 -2 0 2 4 6 1\[S] m M 1\ V IU \L Normal Tubercul osis IHJPAS IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig(7): Arrhen ius plot for S.GOT in both normal and tub er culosis 0 0.5 1 1.5 2 2.5 0.0029 0.003 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 1\T (K) L o g (V ) (I U \L ) Normal Tuberculosi Fif(5):psed o first order plot for S.GOT in both normal and tu ber culosis 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 Time (min) L in (V m a x \V m a x -V ) Tuberculosis normal Fig(6):Hill plot for S.GOT in both normal and Tuber culosis 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Log [S] L o g (V \V m a x -V ) Tuberculosi s Normal IHJPAS 2010) 3( 23 مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد و بعض الدوال الحركیة فى مصول المرضى (GOT) دراسة نشاط االنزیم المصابین بالتدرن الرئوى بروین عبد الصمد اسماعیل، لطفیة محمد حسن ح الدیناالةسام العلمیة، جامعة صال/كلیة التربیة،قسم الكیمیاء االةسام العلمیة،جامعة صالح لدین/قسم الكیمیاء ،كلیة التربیة الخالصة و اس 37بـدى االنـزیم المنقـى اعظـم فعالیـة فـى حـرارةاذ افى المصل GOT فى الدراسة الحالیة تم تنقیة االنزیم ـا ال 7.4الهیــدروجینى تبــادل اال یـــونى الــى فصــل االنـــزیم الــى اربعـــة درجــة و خـــال ل عملیــة التنقیــة ادى كروموتوغرافیــ فى مصول المرضى المصابین بالتدرن الرئوى و اظهرت الدراسة GOTمستویات االنزیم تو قد بحث. اشكال ه فـى المصـول الطبیعیــة ان معـدل نشـاط االنــزیم فـى مجموعــة اذن نشـاط االنـزیم فــى مصـول المرضـى اعلــى مـن معـدال تــ وبینـت (0.65IU/L±27)مقارنـة بالمجموعـة الطبیعیـة والبالغـة (IU/L IU/L 8±90))ت مرضـى التـدرن الرئـوى بلغـ ودرجـة الحـرارة 7.4قمـة نشـاطه فـى اس الهیـدروجینى GOTكمـا ابـدى االنـزیم (P<0.001ارتفاعـا معنویـا 37ć.شـملت ،اذ) الطبیعى و التدرن الرئوى (جین \ولقد ركزت الدراسة على متابعة میكانیكیة التفاعل االنزیمى فى كال النمو لتقـیم اى تغیـر حاصـل فـى عـدد /t1/2)()(وزمـن العمـر النصـفى ((K1)معدالت ثابت التكـوین ، Km,Vmaxقیم (دراسة تغیر معدل سرعة التفاعل االنزیمى من ثم وتركیب المواقع النشطة الموجودة على االنزیم و IHJPAS