1. Comparison of Urease and Diacetylmonoxime in Blood Urea Analysis By By Ogunmolasuyi Adewoyin Martin Department of Biochemistry, Federal University of Technology, Minna, Niger State.

2. Introduction Urea is hydrolyzed with urease (E.C 3.5.15) a nickel dependent enzyme, to yield two molecule of ammonia and carbon (IV) oxide. Urea is hydrolyzed with urease (E.C 3.5.15) a nickel dependent enzyme, to yield two molecule of ammonia and carbon (IV) oxide. In hospital and medical laboratories, it is used for diagnostic purposes, in the determination of urea in biological fluids such as urine and blood. In hospital and medical laboratories, it is used for diagnostic purposes, in the determination of urea in biological fluids such as urine and blood. This work was designed to compare the effectiveness of urease and the conventional diacetylmonoxime in blood urea analysis. This work was designed to compare the effectiveness of urease and the conventional diacetylmonoxime in blood urea analysis.

3. Materials and Methods The urease enzyme was extracted from Mucuna pruriens got from Owo in Ondo State, Nigeria, while five samples of blood were collected from Department of Chemical Pathology, Yusuf Dantsoho Memorial Hospital, Kaduna. Nessler’s reagent, Tris-acetate buffer and all other chemical used were of analytical grade. The urease enzyme was extracted from Mucuna pruriens got from Owo in Ondo State, Nigeria, while five samples of blood were collected from Department of Chemical Pathology, Yusuf Dantsoho Memorial Hospital, Kaduna. Nessler’s reagent, Tris-acetate buffer and all other chemical used were of analytical grade.

4. Urease Extraction Dried Mucuna pruriens was blended coarsely with laboratory mortar and pestle and 50g of the beans meal were soaked overnight in 100ml extraction buffer (0.025M Tris-acetate buffer, pH 6.5) at 40C. The soaked beans meal were swirled for 2mins and then sieved with four layers of pre-washed and dried white muslin cloth, and the filtrate centrifuged (RC-16M Refrigerated Centrifuge Sherwood Medicals England) at15000rev/min for 15mins at 40C. The clear supernatant was collected while the sediment was discarded (Arvind and Nilanjana, 1999).The clear supernatant was fractionated with cold acetone in a range of about one-third the volume of the crude extract at 00C (ice-chest with constant swirling). After 15mins in ice-chest, the suspension (precipitate) was centrifuged at 40C, 15000rev/min for 2mins. There was second addition of acetone of equal volume to the first. The collected precipitate was dissolved in a minimum volume of 0.05M Tris-acetate buffer. Dried Mucuna pruriens was blended coarsely with laboratory mortar and pestle and 50g of the beans meal were soaked overnight in 100ml extraction buffer (0.025M Tris-acetate buffer, pH 6.5) at 40C. The soaked beans meal were swirled for 2mins and then sieved with four layers of pre-washed and dried white muslin cloth, and the filtrate centrifuged (RC-16M Refrigerated Centrifuge Sherwood Medicals England) at15000rev/min for 15mins at 40C. The clear supernatant was collected while the sediment was discarded (Arvind and Nilanjana, 1999).The clear supernatant was fractionated with cold acetone in a range of about one-third the volume of the crude extract at 00C (ice-chest with constant swirling). After 15mins in ice-chest, the suspension (precipitate) was centrifuged at 40C, 15000rev/min for 2mins. There was second addition of acetone of equal volume to the first. The collected precipitate was dissolved in a minimum volume of 0.05M Tris-acetate buffer.

5. Partial Purification of Urease The precipitated protein containing urease activity was loaded onto a sephadex G-100 column (3.0x15cm), previously equilibrated with 0.05M Tris-acetate buffer, pH 7.0 (15ml/g bed volume). After collecting the pass through, the column was washed with two-bed volumes of the same buffer. The adsorbed protein was eluted (20-220ml) using 0.1 M Tris-acetate buffers, pH7.0. Active fractions (100-140ml) were pooled and used. The precipitated protein containing urease activity was loaded onto a sephadex G-100 column (3.0x15cm), previously equilibrated with 0.05M Tris-acetate buffer, pH 7.0 (15ml/g bed volume). After collecting the pass through, the column was washed with two-bed volumes of the same buffer. The adsorbed protein was eluted (20-220ml) using 0.1 M Tris-acetate buffers, pH7.0. Active fractions (100-140ml) were pooled and used.

6. Assay of Urease The reaction mixture contained 0.1M Tris-accetate buffer, pH 7.0, 0.02M urea and the enzyme was incubated (DNP-982 Laboratory incubator; Gulflex Medicals and Scientific England) for 30min at 37oC, and the reaction was stopped by reducing the temperature to 10oC. Urease activity was determined by measuring the amount of ammonia released from urea using the Nessler’s reagent. The reaction was monitored spectrophotometrically (Biochrom (Biochrom Ltd) UV 2800 Double beam; UV/VIS scanning spectrophotometer) at 405nm. The optimum pH, temperature and substrate concentration profile and kinetic parameters ranged from 5.5-8.0, 30-80oC and 0.1-0.6M respectively. The reaction mixture contained 0.1M Tris-accetate buffer, pH 7.0, 0.02M urea and the enzyme was incubated (DNP-982 Laboratory incubator; Gulflex Medicals and Scientific England) for 30min at 37oC, and the reaction was stopped by reducing the temperature to 10oC. Urease activity was determined by measuring the amount of ammonia released from urea using the Nessler’s reagent. The reaction was monitored spectrophotometrically (Biochrom (Biochrom Ltd) UV 2800 Double beam; UV/VIS scanning spectrophotometer) at 405nm. The optimum pH, temperature and substrate concentration profile and kinetic parameters ranged from 5.5-8.0, 30-80oC and 0.1-0.6M respectively.

7. Blood urea Analysis with Urease and DAM To 1.9ml of assay buffer (0.1M Tris-acetate, pH 7.0) was added 1.0ml diluted partially purified urease (PPU) and pre-incubated at 370C. Then 0.1ml serum was added followed by incubation for 30min at 370C. To this reaction mixture, was added cold acetone in appropriate amount (usually one-third) to precipitate the blood proteins. After centrifugation at 5000rev/min for 20mins, an aliquot (1ml) of the reaction mixture was transferred to a 50ml beaker and 1.0ml Nesslers’ reagent was added with constant swirling and the volume made up with distilled water after 1min of reaction. The yellow colour produced was measured at 405nm with UV/VIS spectrophotometer (Biochrom UV 2800 Double beam; UV/VIS scanning spectrophotometer) (Arvind and Nilanjana, 1999). A standard curve with urea concentration in the range of 2.0mM-14.0mM (normal blood urea range is 2.5mM-6.4mM) was drawn under similar condition to estimate the urea concentration of the blood samples. To 1.9ml of assay buffer (0.1M Tris-acetate, pH 7.0) was added 1.0ml diluted partially purified urease (PPU) and pre-incubated at 370C. Then 0.1ml serum was added followed by incubation for 30min at 370C. To this reaction mixture, was added cold acetone in appropriate amount (usually one-third) to precipitate the blood proteins. After centrifugation at 5000rev/min for 20mins, an aliquot (1ml) of the reaction mixture was transferred to a 50ml beaker and 1.0ml Nesslers’ reagent was added with constant swirling and the volume made up with distilled water after 1min of reaction. The yellow colour produced was measured at 405nm with UV/VIS spectrophotometer (Biochrom UV 2800 Double beam; UV/VIS scanning spectrophotometer) (Arvind and Nilanjana, 1999). A standard curve with urea concentration in the range of 2.0mM-14.0mM (normal blood urea range is 2.5mM-6.4mM) was drawn under similar condition to estimate the urea concentration of the blood samples.

8. Diacetylmonoxime (DAM) Test samples StandardBlank Distilled water 1ml1ml2ml Urea acid reagent 2ml2ml2ml Urea colour reagent 2ml2ml2ml 1/100 dil. of sample 1ml--------------- -----------1ml-------

9. Mix the reaction thoroughly and boil for 20mins, cool the reaction and read at 520nm. Mix the reaction thoroughly and boil for 20mins, cool the reaction and read at 520nm. Calculation:Test sample’s Absorbance X Conc. of standard (8.3) Calculation:Test sample’s Absorbance X Conc. of standard (8.3) Standard Absorbance

10. Result and Discussion The results of protein level and specific activity of crude urease and PPU are summarized in Table 2.0. From Figure 1.0 the protein level as well as urease specific activity of the PPU was highest in fraction 120ml. The active fractions 100-140ml was pooled together for blood urea analysis. The results of protein level and specific activity of crude urease and PPU are summarized in Table 2.0. From Figure 1.0 the protein level as well as urease specific activity of the PPU was highest in fraction 120ml. The active fractions 100-140ml was pooled together for blood urea analysis. From the elution profile of sephadex G-100 column chromatography (Figure 1.0), urease was eluted from the column in one peak of protein accompanied with one peak of activity. The protein level of PPU was significantly (p<0.05) reduced when compared to the crude protein. This showed that, gel filtration removed about 78.23% of the contaminating proteins and reduced the protein by 4.60fold. The peak of activity was found in fractions 120ml with urease activity 18.67µmol/s/mg which is 94% and 15.97fold higher than the crude enzyme. El-Shora, (2001) reported on the activity of purified urease from leaves of Chenopodium album. He showed that, the activity increased from 1.6 to 1.9 µmol/s /mg with1.2fold increase. This showed that, Mucuna pruriens contains higher urease activity than leaves of Chenopodium album. These results showed that, with purification there was significant increase in the urease activity. It’s therefore clear that enzyme purification enhances higher catalytic activity which invariably would lead to higher product yield most especially during application. From the elution profile of sephadex G-100 column chromatography (Figure 1.0), urease was eluted from the column in one peak of protein accompanied with one peak of activity. The protein level of PPU was significantly (p<0.05) reduced when compared to the crude protein. This showed that, gel filtration removed about 78.23% of the contaminating proteins and reduced the protein by 4.60fold. The peak of activity was found in fractions 120ml with urease activity 18.67µmol/s/mg which is 94% and 15.97fold higher than the crude enzyme. El-Shora, (2001) reported on the activity of purified urease from leaves of Chenopodium album. He showed that, the activity increased from 1.6 to 1.9 µmol/s /mg with1.2fold increase. This showed that, Mucuna pruriens contains higher urease activity than leaves of Chenopodium album. These results showed that, with purification there was significant increase in the urease activity. It’s therefore clear that enzyme purification enhances higher catalytic activity which invariably would lead to higher product yield most especially during application.

12. The result of optimum pH for crude urease was presented in Figure 2.0, while PPU was assayed at 7.0. This result is supported by other researchers. For instance, optimum pH for urease activity in Jack beans, Rhyzopus oryzea and surface black soil were found to be 7.0, 7.0 and 6.45 respectively. The stability of urease activity at these pH values may be explained by the structural changes occurring in enzyme proteins caused by pH variation. The tertiary structure of a protein depends on interactions such as hydrogen bonding between enzyme amino acids functional groups. A change in pH can alter the ionization of these amino acid side chains and disrupt the native conformation and in some cases denature the enzyme. Hence, each enzyme has an optimal pH range that helps the maintenance of its native conformation in an environment, where it operates. The degree of ionization of the surface functional groups of amino acid residues is a function of the medium’s pH. The main factor involved here is the titration of the ionizable group which maintains surface charge, in the active site, or stabilizes the enzyme resulting in an optimum pH for the enzyme. The result of optimum pH for crude urease was presented in Figure 2.0, while PPU was assayed at 7.0. This result is supported by other researchers. For instance, optimum pH for urease activity in Jack beans, Rhyzopus oryzea and surface black soil were found to be 7.0, 7.0 and 6.45 respectively. The stability of urease activity at these pH values may be explained by the structural changes occurring in enzyme proteins caused by pH variation. The tertiary structure of a protein depends on interactions such as hydrogen bonding between enzyme amino acids functional groups. A change in pH can alter the ionization of these amino acid side chains and disrupt the native conformation and in some cases denature the enzyme. Hence, each enzyme has an optimal pH range that helps the maintenance of its native conformation in an environment, where it operates. The degree of ionization of the surface functional groups of amino acid residues is a function of the medium’s pH. The main factor involved here is the titration of the ionizable group which maintains surface charge, in the active site, or stabilizes the enzyme resulting in an optimum pH for the enzyme.

14. Hence, the application of urease in clinical chemistry for determination of urea in biological fluid such as blood and urine as well as microencapsulation of urease in the development of artificial kidney for those with renal failure is possible, because both blood and urease are stable at pH around neutral. Hence, the application of urease in clinical chemistry for determination of urea in biological fluid such as blood and urine as well as microencapsulation of urease in the development of artificial kidney for those with renal failure is possible, because both blood and urease are stable at pH around neutral.