Microsoft Word - 116-125 116 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 Isolation and Partial Purifiction of Arginase from Sera of Women with Uterine Fibroids   Taghreed U. Mohammd Eiman A. Abass Manar A.Salman   Dept. of Chemistry/ College of Education for Pure Sciences ( Ibn-AL-Haitham)/ University of Baghdad. Received in: 12 January 2015, Accepted in: 25 March 2015 Abstract The first aim of the present study was performed to assay the activity of arginase in sera of women with uterine fibroid.. This study consisted of(50) women with uterine fibroid as patient's group and (30) healthy women as control group. The age ranged between (30-55) years for the two groups. The results showed that highly significant increas (P< 0.0001) in the arginase activity in sera of women with uterine fibroid (7.99± 0.23) I.U/L is found when compared with healthy group (0.52±0.02) I.U/L. The second aim was performed to isolate arginase from sera of women with uterine fibroids. The purification is done by addition of ammonium sulfate, dialysis, gel filtration chromatography by using sephadex G-50 and ion exchange chromatography by using DEAE cellulose A-50. The results showed a single band for isoenzymes following steps by using sodium dodecyl sulfate- polyacryl amide gel electrophoresis (SDS-PAGE) .  Key words: Arginase, Uterine fibroid.   117 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 Introduction Uterine fibroids are benign tumors[1] that develop from the muscle tissue of the uterus[2].They also are called leiomyomas, myomas, fibromyomas, leiomyofibromas. Because the tumor consists of uterine smooth tissue as well as fibroids tissue, the term fibroid does not adequately capture the name of the lesion[3]. These tumors have prevalence ranging from 20% to 40% of the women depending on the age[4]. Studies using both ultra sound and pathologic examination of surgical uterine specimens suggest an overall prevalence of uterine fibroids of over 70%. Uterine fibroids can cause severe problems for women and most of the uterine fibroids do not cause symptoms[5].The size, shape and location of fibroid can vary greatly[6]. They may be present inside the uterus, on its outer surface or within its wall, or attached to it by a stem[7]. A woman may have only one fibroid or many of varying sizes. Some are no bigger than a pea, while others can grow to the size of a melon or larger, when fibroids are diagnosed. The extent of the disease is determined by comparing the size of the uterus to typical size during pregnancy, for example a large fibroid or multiple fibroids may enlarge the uterus to the same as a six or seven month pregnancy[8]. Arginase (EC.3. 5. 3.1) belongs to the urea hydrolase family which catalyzed the fifth and final step in the urea cycle, converting L-arginine into L-ornithine and the urea[9]. In most mammals, two isozymes of this enzyme exist; the first, Arginase I. functions in the urea cycle, and is located primarily in the cytoplasm of the liver. The second isozyme, Arginase II, has been implicated in the regulation of the arginine/ornithine concentrations in the cell. It is located in mitochondria of several tissues in the body, with most abundance in the kidney[10] ,prostate, testis ,epididymis, seminal plasmavesicles ,prostate and human sperm cells. It may be found at lower levels in macrophages, lactating mammary glands and brain[11]. The second isozyme may be found in the absence of other urea cycle enzyme[12] . The first aim of this study was performed to measure arginase activity in sera of women with uterine fibroids comparing to healthy women and the seconed aim was performed for partial purification and isolation of arginase isoforms from the serum of women with uterine fibroids. Material and Methods Subjects  The study is conducted during the period from 1st Dec. 2013 to the end of May 2014 at obstetrics and gynecology deportment in Baghdad city (Baghdad Teaching Hospital and Al- Yarmuk Teaching Hospital). The study included (50) women with uterine fibroids which were diagnosed by ultrasound and healthy group consisted of (30) women as control group. The age of the two groups ranged (30-55) years. Specimens Collection and Analysis Venous blood samples were drown from each patient then transferred immediately to a clean dry plain tube. After removing the needle, the blood was allowed to clot for at least (10-15) min, at room temperature and then centrifuged for (15) min, at (3500 rpm). Serum is removed and assayed immediately and the samples were stored at 4°C for the purpose of conducting the required measurements. 118 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 Methods  1-Assay of Arginase Activity in Serum Arginase activity was measured in serum according to (Coulomble., 1963)method [13]. Enzyme unit: is the amount of enzyme that catalyses the reaction of 1µmol of substrate per minute.   2- Estimation of Protein Concentration The total protein concentration was estimated in blood serum using a kit provided from a company (SPINREACT) depending Biuret method. (Tiets,N. W. et al., 1995) [14]. 3- Purification of Arginase from Serum of Women with Uterine Fibroid Enzyme purification process had been done using the following steps: Precipitation by Using Ammonium Sulfate of Concentration 35% The most frequent used method of protein precipitation is adding of non organic salts like ammonium sulfate, or potassium phosphate. Large amount of water molecules will be bonded to ammonium sulfate when the protein is dissolved in water leading to decrease the amount of water molecules which are interferes with protein, therefore, protein molecules will be concentrated with each other and precipitate [15]. A solid state ammonium sulfate of (0.7gm) was added gradually to a (4mL) serum in a beaker with constant stirring at about (4°C) for one hour till the solution became turbid. Then it was put in a centrifuge at speed (4000 rpm) for (20 min) to split the precipitate from the leach. Finally the precipitate was dissolved in a less amount of buffer (MnCl2, Tris- HCl pH 8.4).Activity and protein concentration were measured.   Dialysis  Dialysis process had been done by putting the protein solution prepared in the previous step into a tightly wrapped cellophane bag from bottom and then it was wrapped tightly also from its top then the pipe was left in to a container which contains the buffer solution (Na2CO3 (0.1M), MnCl2 (0.005M), Tris-HCl (0.005M)), this process was done at (4°C) with constant stirring using magnetic stirrer. After finishing the dialysis process the final volume of the resultant solution was calculated . The amount of protein and enzyme activity were estimated.     Gel Filtration Four mL serum taken from dialysis bag was added slowly on the surface of the sephadex G-50 column (20x1.5 cm) and left for five minutes to be absorbed. Ten fractions were collected by bassing buffer solution (Na2CO3 (0.1M), MnCl2 (0.005M), Tris-HCl (0.005M) pH 8.3) through the column .Entire operation was carried out inside a refrigerator. Flow rate ( 2 mL/min). Ion Exchange Chromatography Two mLs of the fresh filtred serum sample were passed through a column of sephadex ion exchange chromatograghy DEAE-Cellelose A 50 pre-equilibrated (Na2CO3 (0.1M), MnCl2 (0.005M), Tris-HCl (0.005M) pH 8.3) . The sephadex ion exchange column of the size of (20x1.5 cm) containing gradient concentration of potassium chloride (0.1- 0.3M/L). Entire operation was carried out inside a refrigerator. Flow rate ( 2mL/min). 119 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 SDS- PAGE Electrophoresis SDS- polyacrylamide gel electrophoresis (10%) was carried out according to Laemmli et al [16]. The purified enzyme was treated with 1% SDS and β-mercaptoethanol for 10 mintues at 100ο C and loaded in wells. Samples stacking was done at 10 mM and resolution was carried out at 15 mA constant current. Molecular weight standards were bovine serum albumin (66,000), ovalbumin (45,000), chymotrypsinogen (25,000) (Sigma). After electrophoresis gel was stained in 0.25% w/v Coomassie brilliant blue (R-250) prepared in 50% v/v methanol and 10% v/v acetic acid. The gels were destained by passive diffusion of dye in 50% v/v methanol and 1% acetic acid after changing the destaining solution for 2-3 times. Statistical Methods Statistical analysis was used to show the mean and standard error deviation of variables. The significance of difference between mean values was estimated by student T-Test . The probability p<0.05= significant . p>0.05 = non- significant. ANOVA test was used to show the differences between variables of differentiated groups. The data were processed with the software package SPSS ( statistical package for social sciences ) Ver.17 and Microsoft Excel version 2007 . Results and Discussion Table (1) shows the mean±SE and p- value of the arginase activity in sera of women with uterine fibroid and healthy women. The result in table (1) shows a highly significant increase in the arginase activity in the women with uterine fibroid (7.99 ± 0.23 IU/L) when compared to healthy women (0.52 ± 0.02 IU/L). Arginase can redirect the metabolism of L-arginine in smooth muscle cells from nitric oxide to L-ornithine and the formation of polyamines and L-proline, which can induce vascular lesion formation by stimulating smooth muscle cell proliferation and collagen deposition. These actions of arginase are further magnified by the suppression of NO release, which serves as a well- recognized inhibitor of smooth muscle cell growth and collagen synthesis[17]. While (Kaplen et al.,2012)[18], revealed the increase in arginase activity may limit nitric oxide synthase (NOS) activity and lead to decrease of the inhibitor effect on xanthine oxidase activity. In this case, it results in more superoxide radical production and tissues damage. As evidenced by the up regulation of arginase in specific disease states, its distribution in vagina, and its modulation by sex steroid hormones, this enzyme may also tissue growth fibrosis, and immune function[19]. The recent study demonstrated that arginase activity may be attributed to change in permeability of fibroid cell as a result of many biochemical changes in cell surface, that included changes in the appearance of cell surface such as variation in glycolipid and mucin[20]. Partial Purification of Arginase from Sera of Women with Uterine Fibroids Table(2) summarises the result of isolation and partial purification of arginase and its isoenzymes from women sera with uterine fibroids which carried out through steps. In the first step, the proteins precipitate by using ammonium sulfate salt in concentration of (35%) for the concentration of enzyme and purified, degree of purity was obtained (9.9) fold and excess salt was eliminated during dialysis process by Na2CO3 (0.1 M), MnCl2 (0.005 M), Tris.HCl (0.005 M) with pH= 8.3. The degree of arginase purity reached to(3.337) fold in this stage with enzyme yield (92.46%) . The third step included purification of enzyme by gel 120 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 filtration using (Sephadex G-50) which the degree of arginase purity reaches to (5.822) fold which enzyme yields (47.60%) in this stage ,as shown in figure (1).    The purification by ion exchange chromatography is among the significant approaches to isolate and purify the enzyme that is based on the charge difference so the enzyme was purified by using DEAE-cellulose A50. Four isoenzymes were obtained as illustrated in figure (2) and variant purity degree as the degree of purification of I (4.407) fold and II (4.867) fold for the isoenzymes III, IV (5.834), (5.696) folds respectively, as illustrated in table (2). The literatures indicate that two isoenzymes were separated from adult human's liver by using CM-cellulose chromatogarphy [21] and the two isoenzymes of the arginase from rat's liver [22] by using DEAE-cellulose. Tarrab et al., 1974) isolated three isoenzymes from the rat's liver using CM-cellulose chromatography[23]. Gel electrophoresis was used to confirm the purity of the isolated isoenzymes by using sodium dodecyl sulfate- polyacryl amide gel electrophoresis (SDS-PAGA) which appeared as single band. Conclusion These data suggest that an isolation and partial purification of isoenzymes of arginase from sera of women with uterine fibroids by gel filtration and ion exchange chromatography four isoenzymes were obtained (I,II,III,IV).   References   1. Leppert, P.; Fouany, M.; and Segars, J. H. (2013). "Fibroids". 1stEd. John Wiley & Sons, Ltd.pp 1-11. 2. Nivethithai, P.; Nikhat, S. R.; and Rajcsh, B. V.( 2010)." Uterine fibroids".A review. Indian J. Pharmact., 3(1): 6-11. 3. Levy, B.; Mukherjee, T.;and Hirschornk. (2000). Molecular cytogenetic analysis of uterine leiomyoma and leiomyosarcoma by comparative genomic hybridization. Cancer Genetcytogenet., 121: 1-8. 4. Duhan, N.;and Sirhiwal, D. (2010). Uterine myomas revisited. Eur. J. Obstet Gynecol. Repro. Biol., 152: 119-25. 5. Lgnacio; E. A.; and Venbrux, A. C. (2012). Women's health in interventional radiology, 5th Ed. Springer., 4-6. 6. Acton, Q. A.( 2012). Leiomyomas: New insights for the healthcare professional, 2012th Ed. Scholarly, 2. 7. Goodwin, T. M.; Montaro, M. N.; Muderspash, L.; Paulson, R.;and Roy, S. (2010) . "Management of common problems in obstetrics and gynecology", 5th Ed. Wiley& Sons.291-292. 8. Ficalora, R. D. (2013) . "Myoclinic internal medicine board review", 10th Ed. Oxford University Press.712-714. 9. Nelson, D. L.;and Cox, M. M. (2013) . "Lehningerprinciples of biochemistry" , 6th Ed. W. H. freeman & Company, 704-706. 10. Stephen, D. C.; Hong, Y.; Wayne, W.G.; Rita, M. K.; Paul, Y.;and Ramaswamy, K. I. (2004) . Arginase I and II: do their functions overlap?. Mol. Gen. Metab., 81: 38-44. 11. Elgün, S.; Kacmaz, M.; Sen, I.and Durak, I. (2000) . Seminal arginase activity in infertility. Springer, 28: 20-23. 121 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 12. Di Costanzo, L.; Moulin, M.; Haertlein, M.; Meilleur, F.and Christianson, D. W. (2007) . Expression, purification, assay, and crystal structure of perpetuated human arginase I archives of biochemistry and biophysics, 465(1): 82-9. 13. Coulomble, J. J.;and Favreau, L. (1963). A new simple semimicro method for colorimetric determination of urea.Clin. Chem., 9-102. 14. Tiets, N. W. (1995) . "Clinical guide to laboratory".Tests, 3th Ed. AACC. 15. Robyt,J.F.; and White,B.J.(1987) . Biochemical Techinques: Theory and practice, Books/cole publishing company, Belmont,CA. 16. Laemmli, U. K. (1970) Cleavage of structural proteins during the assemble of the head of the bacteriophage T4. Nature., 227:680-685. 17. Durante, W.; Johnson, F. K. and Johnson, R. (2007). Arginase: A critical regulator of nitric oxide synthesis and vascular function. Clin. Exp. Pharmacol. Physiol., 34(9): 906-911. 18. Kaplan, I.; Aydin, Y.; Bilen, Y.; Gen, C. F.; Keles, M. S.;and Eroglu, A. (2012) . The evaluation of plasma arginine,arginase, and nitric oxide levels in patients with esophageal cancer, 42(3): 403-409. 19. Kim, N. N.; Christianson, D. W.;and Traish, A. M. (2004). Role of arginase in the male and female sexual Aromsal.Response. J. Nutr., 134(10): 2873-2879. في مصل RNase .ز . دراسة كيموحيوية ألنزيم الرايبونيوكلي٢٠١٣، دعاء حسين. ؛عمرالعباجي، صبا زكي .٢٠ .٨٠-٦٤): ٥(٢٤بأورام الرحم. مجلة علوم الرافدين، وأنسجة المصابات دم 21. Bascur, L.; Cabello, J.; Veliz, M.;and Gonzales, A. (1970). Biochim.Biophys.Acta., 128: 149-154. 22. Gasiorowska, I.; Porembska, Z.; Jachimowic, Z. J.;and Machnacka, I. (1970). Acta.Biochim. Pol., 17: 19-30. 23. Tarrab, R.; Rodriguez, J.; Huitron,C.; Palaciros, R.; and Soberon, G. (1974) . Eur. J. Biochem., 49: 457-468. 122 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 Table No. (1): mean±SE of arginase activity in the sera of women with uterine fibroid and healthy women. p-value mean± SE N Arginase(I.U/L) <0.0001 7.99 ± 0.23 50 Uterine fibroid women 0.52 ± 0.02 30 Healthy women   Table No. (2): Isolation and purification of arginase isoenzymes from uterine fibroids women. Step Elute (mL) Activity I.U/L Total activity (IU) Prot Conc. mg/L Total port (mg) Specific activity IU/mg Purifi- cation Fold Yield % Crude serum 4 7.30 0.0292 840 3.360 0.00869 1 100 Ammoniu m sulphate 2 9.9 0.0198 529 1.587 0.0187 2.151 67.80 Dialysis 2 13.94 0.027 480 1.440 0.0290 3.337 92.46 Sephadex G-50 2 6.99 0.0139 138 276 0.0506 5.822 47.60 Ion- exchange Iso.I 2 4.6 0.00920 120 240 0.0383 4.407 31.50 Iso.II 2 5.21 0.0104 123 246 0.0423 4.867 35.63 Iso.III 2 5.99 0.0119 118 236 0.0507 5.834 40.75 Iso.IV 2 5.8 0.0116 117 234 0.0495 5.696 39.72   123 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015   Figure No.(1):Purification of arginase from uterine fibroids women by using chromatography Sephadex G-50.   Figure No. (2): Isolation of arginase isoenzymes from srea uterine fibroids women by using chromatography Ion exchange DEAE- cellulose A50.   0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A ct iv it y a rg in a se ( I. U /L ) Fraction No. Activity ((I.U/L) Protein Concentration (g/dL) 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 6 7 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 A ct iv it y a rg in a se ( I. U /L ) Fraction No. Activity ((I.U/L) Protein Concentration (g/dL) 124 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015                     Figure No.(3):Electrophoresis of isolation arginase 125 | Chemistry ٢٠١٥) عام 2(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28(2) 2015 ألنزيم األرجينيز من أمصال النساء المصابات جزئيةوتنقية فصل بتليف الرحم تغريد علوم محمد أيمان عبد علي عباس منار عباس سلمان جامعة بغداد –) أبن الھيثم الصرفة)كلية التربية للعلوم /قسم الكيمياء ٢٠١٥ ر: اذا٢٥البحث في ل، قب٢٠١٥كانون الثاني ١٢استلم البحث في: الخالصة تضمنت ھذه .ھدفت الدراسة الحالية أوال إلى قياس فعالية أنزيم األرجينيز من امصال النساء المصابات بتليف الرحم ) امرأة سليمة بوصفھم مجموعة مقارنة، تراوحت أعمارھن بين 30) امرأة من المصابات بتليف الرحم و (50الدراسة ( )في فعالية أنزيم األرجينيز في النساء P>(0.0001 ) سنة في المجموعتين. بينت النتائج وجود ارتفاع معنوي30-55( ) وحدة عالمية/لتر في 0.52±0.02) وحدة عالمية/لتر، بينما كانت معدل فعاليته (7.99±0.23المصابات بتليف الرحم ( األصحاء. انزيم االرجينيز من امصال النساء المصابات بتليف الرحم من خالل الترسيب اما الھدف الثاني ھو فصل وتنقية متناظرات وكروموتوغرافيا التبادل sephadex G-50بكبريتات االمونيوم والديلزة وبأستعمال تقنية كروموتوغرافيا الترشيح الھالمي وعند استعمال طريقة الفصل تم فصل أربع متناظرات أنزيمية تختلف في درجة تنقيتھا. DEAE cellulose-50األيوني ظھرت حزمة Coomassie blue R250وبأستعمال صبغة %10بتركيز SDS-PAGAعلى ھالم بالترحيل الكھربائي منفردة لكل متناظر. .: أنزيم األرجينيز، تليف الرحمالكلمات المفتاحية