IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 The Study o f Serum GOT(Glutamic Oxalacetic Transaminase) Activity and So me kinetic Parameters in Patient with Pulmo nary Tuberculosis P. A.Ismahil , L. M.Hassan Departme nt of Chemistry ,College of Education Science,Unive rsity of Salahaddin Abstract In the p resent st udy , serum GOT(Glutamic Oxalacetic Transaminase) was p urified, the p urified enzy me showed the maximum activity at 37ºC and p H 7.5. During p urification of serum GOT ion exchange chromatography lead to four sep arate forms (termed I, II, III and IV). GOT II with the highest sp ecific activity was p ure after chromatography on Sephacry l S- 300. . S.GOT levels were invest igated in serum samples from p atient with p ulmonary tuberculosis .The S.GOT levels were determined at and 37ć .The st udy revealed that the serum GOT activity was higher in p atient of tuberculous p ulmonary than in control subjects.T he mean serum GOT activity in the p atients 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 comp rehensive measurement of the rate reaction kinetics(Km,Vmax,Hill coefficient),the first order forward rate constant k1 and half life t1/2 of the enzy me reaction in both normal and tuberculosis serum to evaluate any change In the number and st ructure of the active site then the rate of enzy me –substrate binding. Key words :Glutamic O xalacetic Transaminase.Tuberculosis Introduction Tuberculosis is a chronic bacterial infection caused by M y cobacterium tuberculosis and characterized by the formation of granulomas in infected tissue and by cell –mediated hy p ersensitivity . 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 sp an of time become a major health p roblem in the third word. The severity of the diseases can be judged by the fact that it affects all ages [4,5,6].M y cobacterium tuberculosis is resp onsible for more morbidity in humans than any other bacterial diseases.M y cobacterium infects 1.1 billion p eople p er year which is equal to 33% of the entire word p op ulation [7,8,9] Glutamic oxalacetic transaminase is a tissue enzy me that catalyzes the transfer of amino and keto group between alp ha-amino acid and alp ha-keto acid .The GOT exist in the tissues of many organs .Necrotic activity in these organs causes a release of abnormal quantities of enzy me 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 t y p es 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] Experime ntal *S amples collection and S eparation Blood analysis is usually done on venous of cap illary blood.60 samples of the blood were through out the investigation of GOT enzy me activity ,35 of these samp les 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 st anding at room temp erature until it has clotted. Restriction of the clot may be assist ed by gentle loosing it from the walls of the container. The serum was sep arated 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 p arched from BDH ,FLUKA *Instruments and Apparatus -Sepctrop hotometer, sup ertonic 601 -Philip s p H-meter -Thermost atic water bath -Centrifuge T.5 *Methods A- Esti mation of S.GOT activity S .GOT activity was determined by measurement of oxaloacetate p roduced during the assay according to the method of Bergmey er and Bernt (1965). The samples were incubated at 37◦C for 1 h in tubes containing 100 μM asp artic acid, 2μM α-ketoglutaric acid, 20mM sodium phosp hate buffer, pH 7.5 and app rop riate amount of enzy me to give a final volume of 1.0ml. The reaction was st op p ed by the addition of 1ml 2,4-dinitrop henylhydrazine reagent and allowed to incubate 20min at room temp erature and 10ml 0.4N sodium hy droxide was added. The absorbance was recorded at 505 nm. The enzy matically liberated oxaloacetate was calculated from a st andard curve absorbed at 510 nm ,using series concentration of substrate[S] Fig(3). Purifi cation of aspartate transaminase S tep 1: Freshly drawn, heparinized human blood is centrifuged at 5000 rp m for 30 min, and the p lasma is treated with ammonium sulfate. The protein fraction p recipitating between 30 and 80% saturation is collected S tep 2: The p ooled sup ernatants (630ml) were p laced in a 60°C water bath for 40min with frequent mixing and then centrifuged at 100OOg for 30min. S tep 3: The sup ernatant from st age 2 was further p urified by (NH4)2SO4 p recipitation. Op timum precipitation occurred between 64 and 72% saturation, with an overall p urification factor of 69 and a y ield of 37%. and a y ield of 30% were obtained. Prot ein p recipitating overnight in the op timum range was collected and redissolved in 0.01 M -sodium p hosp hate buffer, pH7.4, dialy zed against the same buffer S tep 4:DEAE- cel lulose chromatography The dialyzed sup ernatant from Step 3 was app lied directly to a DEAEcellulose column (20×2.6 cm i.d.) equilibrated with 50mM sodium p hosp hate buffer, p H 7.5. The adsorbed material was eluted with a st epwise gradient ranging from 0 to 0.4M NaCl p repared in the same buffer at a flow rate of 60ml/h and 10ml fractions were collected. Prot ein fractions exhibitingAST activity were eluted with 0.0, 0.05, 0.1 and 0.2M NaCl, resp ectively and designated AST I, II, III and IV, according to elution order. S tep 5: Se phacryl chromatography AST II was app lied to a Sephacry l S-300 column (95×1.6 cm i.d.) equilibrated with 50mM sodium p hosp hate buffer, p H 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 Prot ein was determined either by measuring the absorbance at 280 nm (Warbur g and Christian, 1942) or by the method of Bradford (1976) using bovine serum albumin as a st andard IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 B-Esti mation of physi cal constant (Km& Vmax) value s The values of Km &Vmax was determined in normal and tuberculosis cases the same exp erimental p rotocol of activity was app lied 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 lott ing data .Theses were of 1-M ichaeles-M entin p lot in which the rate of reaction(V)is p lott ed Vs [S],directly 2-Lineweaver-Burk p lot a reciprocal plot of 1/V Vs 1/[S] has been app lied C-Esti mation of the rate association constant (K1),hal f-life time (t1/2) Following time course reaction ,the order of the enzy matic reaction (K1) and half life times(t1/2) were determined .The same p rotocol of the activity was ap p lied at const ant [S] but the time of incubation was varied (fig5,6) D-Esti mation of the activation Ene rgie s(Ea*) The same p rotocol of GOT determination was adop ted and the only difference is the use of various incubation temperature (fig7) Results and Discussion The p urification of AST is summarized in Table 1. From the elution p rofile 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, resp ectively. Furt her p urification was rest ricted to GOT II. A Sephacry l S-300 column (Figure1b) was used to obtain GOT II. with the highest p ossible sp ecific activity (3.63 units/mg p rotein). The activity of GOT in tuberculosis was estimated quantitatively and compared with that of normal using developed calorimetric method .Data obtained revealed sp ecific elevation of GOT activity (90±8.1 IU/L) in case of tuberculosis fig(2) comp aring with normal individuals (27±0.65 IU/L) .This is due to that serum GOT level p ositively corrected to the diseases extent in tuberculosis of immunocomptenl p atients .The presence of elevated enzy me activity in the p lasma may indicate tissue damage accomp anied by increased release of intracellular enzy mes. M any diseases that cause tissue damage result in an increased release of intracellular enzy me in to the p lasma .The level of sp ecific enzy me activity in the p lasma frequently correlates with the extent of tissue damage. Thus ,the degree of elevation of a p articular enzy me activity in p lasma is often useful in evaluating the p rognosis of the p atient [15].The Km values for the serum GOT decreased in tuberculosis p atient( 0.64 ±0.5mM )fig.4 table2) as comp ared to normal group (1.03 ±0.8mM ) .This means that the affinity of the enzy me 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 p atients (90±8 IU/L) (IU/L) (fig 3 table2) as comp ared to normal(27±0.65 IU/L) these results are accep table because there is an increase in lymphocytes and macrophages activities and concentration in tuberculosis p atient 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 enzy me reaction in both normal and tuberculosis p atients reaction are of the p seudo first order ty p e 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 p atient but (t1/2) decreased as comp ared with normal group (fig 6 table3), so one can conclude that the diseases may affect equilibrium constant(Keq) of (Ea*)comp lex formation through out increasing the association const ant [18,19]. IHJPAS IB N AL- HAITHAM J. FOR PURE & APPL. SC I VO L. 23 (3) 2010 The results also showed that the tuberculosis affected (decreased) the half life time of serum GOT enzy me 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 enzy me binding site to their substrate was estimated from hill p lot fig6.The results (table3) revealed that there were no cooperation and no significant change in (n)value in the case of tuberculosis comp aring with that of normal group s.( Fig7 table4) rep resent the Arrhenius p lot for the serum GOT enzy me in normal and tuberculosis p atient from the figures the energy of activation of the enzy me substrate reaction were estimated (table4).The results show that (Ea*)for enzy me reaction decreases in tuberculosis case .These results indicate that the diseases may affect the mechanism of the enzy me reactions, this p redicts that there is another p athway for the enzy me-substrate comp lex((Ea*)[20] IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I 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)"T ransmission 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- oxo glutaratetransfesrase chromatographi purification and cryst allization of the enzyme 44-:317from seed of glycine hisp ida var ch eep ear"Rev.Roum.Biochem. 13-Tonhazy ,N. ;White, N. and Umbreite, W.(1960) "a rapid method for t he est imation of the glutamic-asp artic transaminase in tissue and its app lication of radation." Arch. 42-6:.328Biohem. 14- Bergmey er, H.U. and Bernt, E.(1965). Glutamate oxaloacetate transaminase. In: M ethod of enzy matic analysis, H.U. Bergmey er (ed.), p p . 837–853. Acad p ress, New York. 15-Chmpe,P.C.and Harrvey ,R.A. (1994)"Lipp incotts illustrated ".Reviews Biochemist ry .2 nd ed.lipinott .J.B. USA IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL. 23 (3) 2010 ed th ,52Varly ,H.;Alan,H. and M aurice,B.(1980)."p ractical clinical ch emistry ,Vol -16 .Willam Hein mann M edical Books LTD .London 17-Gyt on,A.C.(1991) "Text book of medical physiology 8 th ed .W.B.Saunders.USA 18-Ross,E. ;M aguire,M . and Stugll,T.(1977)."Relationship between the β-adrenergi 75-:576252receptor and adenylate cy clase.J.Biol.Chem. 7407-:7736254Waelbo eck,M .;Vanobber gh, E. and Demeyte ,D.(1979) J.Biol.Chem. -19 1789-:177366Nemthy ,G. and Cheraga, T.(1972) J.Phy s.Chem -20 IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Table (1): Purification sche me for serum GOT II Recovery % Fold purification Speci fic activity(units/ mg) protein T otal protein mg T otal units Purific ation 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 p roducing 1 μmol oxaloacetate (pyruvate) p er h under st andard assay conditions. Table(2):Km and Vmax value of Normal& Tuberculosis S GOT Normal Tuberculosis Enzy me Vmax(IU/ L) Km(mM ) Vmax(IU/ L) Km(mM ) GOT 27 1.03 90 0.64 Table(3):Enz yme reaction parameters of Normal and Tubercul osis S GOT Hil Coefficient (n) t1/2 K1(min-¹) Enzy me 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 e nergy of Normal and Tubercul osis S GOT Activation energy Ea Enzy me Tuberculosis Normal 7925.18 8882.18 GOT Fig. (1) (a): A typical e luti on profile for the chromatography of. S erum GOT on DEAE- cel lul ose column (20×2.6 cm i.d.) previously equi librated with 20mM sodium phosphate buffe r, pH 7.5 at a flow rate of 60 ml/h and 10ml fractions. (b) A typical eluti on profil e for the chromatography of S erum GOT II DEAE-cel lul ose fraction on S ephacryl S -300 column (95×1.6 cm i.d.) previously equi librated with 20mM sodium phosphate buffe r, 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):Ser um GOT l eve ls in norm al and tuber cu losi s gr oup 0 20 40 60 80 100 1 2 S .G O T a c ti v it y ( IU \L ) No rm al Tub er cul osis 27 90 Fi g(3):Michae lis- M ente n plot for both no rma l and tuber culosi s 0 20 40 60 80 100 120 0 1 2 3 4 5 6 [S]m M V I U /L Tubercul osis Normal Fig(4): Linw e ave r-Bur k pl ot S. GOT for both tuber culosi s and nor m al se r um -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 I U \L Normal Tuberculosis IHJPAS IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (3) 2010 Fig(7): Arr hen ius plot for S.GOT in both n or mal an d 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 firs t o rd er plo t for S.GOT in both nor m al an d tu ber cul osis 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 Fi g(6):Hill plo t for S.GOT in both nor mal and Tu ber cu losi s 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 L og [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 تبــادل اال یـــونى الــى فصــل االنـــزیم الــى اربعـــة درجــة و خـــال ل عملیــة التنقیــة ادى كروموتوغرافیـــا ال 7.4الهیــدروجینى فى مصول المرضى المصابین بالتدرن الرئوى و اظهرت الدراسة GOTمستویات االنزیم تو قد بحث. اشكال ان معـدل نشـاط االنــزیم فـى مجموعــة اذن نشـاط االنـزیم فــى مصـول المرضـى اعلــى مـن معـدال تــه فـى المصـول الطبیعیــة 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