Studies on separation and properties of lumbrokinase in Pheretima praepinguis

The purpose of this study was to separate lumbrokinase in Pheretima praepinguis and examine its enzymatic properties. With P. praepinguis as material, lumbrokinase was separated with the salting out method. Lumbrokinase activity was measured with casein medium plate method. Effect of pH and temperature on lumbrokinase activity was studied. Results of lumbrokinase separated from P. praepinguis was relatively high. Lumbrokinase activity in neutral or slightly alkaline environment was higher. Lumbrokinase had tolerance ability to high temperature, with highly enzymatic activity under 60°C and wide range of temperature adaptation. Article Info Received: 10 January 2016 Accepted: 8 February 2016 Available Online: 6 March 2016 DOI: 10.3329/bjp.v11iS1.26707 Cite this article: Mei T, Cao L, Zi L, Mingfu G, Die H. Studied on separation and properties of lumbrokinase in Pheretima praepinguis. Bangladesh J Pharmacol. 2016; 11: S106-S109. Studies on separation and properties of lumbrokinase in Pheretima praepinguis Tang Mei, Liu Cao, Liang Zi, Gong Mingfu and Hu Die College of Life Science, Leshan Normal University, Leshan, Sichuan 614 000, China. This paper was presented in the 3rd International Conference on Biomedicine and Pharmaceutics in Zhuhai, China, on December 11-13, 2015. worms. The surface moisture was absorbed with the filter paper. The weight was taken and then added two volumes of phosphate buffer (pH 7.8). The tissue homogenizer was used to homogenize for 5 min at 4°C. Then the sample was kept standing overnight at 4°C. Finally the sample was centrifuged at 5,000 rpm for 10 min and discarded the pellet. The solid ammonium sulfate was added to the supernatant until saturation. The sample was again centrifuged at 4°C, 40000 rpm for 10 min. The precipitate collected was dissolved in a phosphate buffer solution, dialyzed with a dialysis bag and then desalted using a freeze dryer and freeze-dried to obtain a powdery substance of a large number of light-brown. The crude product was lumbrokinase, stored at 4°C temperature (Astrup and Mullertz, 1952). Preliminary determination of lumbrokinase activity The initial activity of lumbrokinase preform was determined by Casein plate method (Astrup and Mullertz, 1952). The size of diameter of the transparent circle represents casein hydrolyzing activity of lumbrokinase. Determination of the activity of lumbrokinase by casein as substrate Casein powder (1 g) was added to Tris-HCl buffer (volume: 100 mL, 0.05 mol/L, pH 7.8) and lysed by heating, so that the substrate concentration was 1%. It was incubated at 37°C. 2 mL of lumbrokinase treated enzyme solution was drawn, quickly added 2 mL 37°C pre-insulated substrate solution, shake well, continue to incubate for 30 min, and finally added 2 mL of 10% trichloroacetic acid solution to stop the reaction. The sample was kept at room temperature for 30 min, filtered and then the absorbance of filtrate was determined at a wavelength of 280 nm. Determination of optimum pH of lumbrokinase activity Taken lumbrokinase freeze-dried powder into enzyme solution, take 1 mL of enzyme solution were added 2 mL pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 phosphate buffer, mix. After 4 hours at room temperature, the absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated to determine the optimum pH of their lumbrokinase activity. Determination of optimum temperature and thermal stability of lumbrokinase 2 mL lumbrokinase enzyme solution and 2 mL 1% concentration of casein solution respectively incubated for 10 min at different temperatures including 20, 30, 40, 50, 60, 70, 80, 90°C. Then the enzyme solution mixed with the casein solution at same temperature. Mixed solution was incubated for 30 min at the original temperature. Finally, 2 mL 10% trichloroacetic acid solution was added to the mixed solution to stop the reaction. The mixed solution was filtrated after standing at room temperature for 30 min. The absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated, to determine the optimum temperature. Two milliliters of enzyme solution was incubated at the optimum temperature conditions for different time including 2, 4, 6, 8, 10, 12, 16 and 24 hours. Then the enzyme solution was mixed with casein solution (1%; 2 mL). The mixed solution was incubated for 30 min at the original temperature. Finally, 2 mL 10% trichloroacetic acid solution was added to the mixed solution to stop the reaction. The mixed solution was filtrated after standing at room temperature for 30 min. The absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated, to determine the holding time of lumbrokinase activity under optimum temperature. Results Crude extracts and active detection of lumbrokinase Total 12 g freeze-drying crude product of lumbrokinase was obtained crude extract by ammonium sulfate salting precipitation method with 2053.1 g clean fresh P. praepinguis. Lumbrokinase yield was 0.5% which was relatively low. Lumbrokinase activity was higher and the average diameter of the transparent circle was 15 mm with casein plate method (Figure 1). Determination of optimum pH of lumbrokinase activity Lumbrokinase activity was significantly affected by pH (Figure 2). Lumbrokinase activity was relatively stable Bangladesh J Pharmacol 2016; 11: S106-S109 S107 Figure 1: Lumbrokinase activity detection with casein plate method and high activity in the alkaline environment (pH 7-9), Lumbrokinase activity retention rate maintain 75% when pH increased to 11. Lumbrokinase activity gradually reduced in an acidic environment with pH decreased, lumbrokinase activity retention rate was less than 25% at pH 4 and zero at pH 3. Effect of temperature on lumbrokinase activity Lumbrokinase activity was significantly affected by pH (Figure 3). Lumbrokinase activity retention rate was 100% below 60°C, rapid declined less than 10% when temperature raised 70°C and 0% when temperature reached 80°C. Lumbrokinase activity kept intact and lumbrokinase activity retention rate maintained 100% in temperature range of 20°C to 60°C, but the water bath time of enzyme solution at 60°C also affected the lumbrokinase activity (see Figure 4). Lumbrokinase activity retention rate maintained 100% with water bath 60°C, 2 hours. With the water bath time in 60°C extended, lumbrokinase activity decreased gradually and stabilized with water bath 60°C, 16 hours. Lumbrokinase activity retention rate stabled at around 33.0% after 16 hours of water bath in 60°C.


Introduction
Earthworm fibrinolytic enzyme, also known as lumbrokinase (Mihara et al., 1983), is a set of homoserine proteolysis enzyme with fibrinolytic activity derived from earthworm.With a strong anti-thrombotic and thrombolysis effect, lumbrokinase can effectively degrade fibrinogen thereby reducing blood viscosity (Zhu et al., 2009;Li, 2009).Applications of lumbrokinase in the field of heart and blood system diseases is very extensive because of good effect for the treatment of cerebral ischemic diseases (Qian et al., 2005;Yu et al., 2009), cardiovascular disease (Wei, 2010), diabetes (Hang, 2009;Chu et al., 2008) etc. Lumbrokinase may have multiple single component (Chu et al., 2008;Wang et al., 2003), the enzyme activity is affected by many factors (Wang et al., 2003;Chi et al., 1999;Zhou et al., 2011).
In this experiment, crude products of lumbrokinase was extracted from P. praepinguis grew in Mount Emei.Plasmin activity of crude lumbrokinase was detected by using casein plate method (Wu and Zhao, 2004).To seek broader way of lumbrokinase biological preparation, the effects of pH and temperature on lumbrokinase activity was been further studied.

P. praepinguis
P. praepinguis was collected from the cool, moist, humus -rich soil altitude 500-700 meters from Baoguo Temple to Qingyin pavilion in Mount Emei, Sichuan Province, China.

Preparation of crude products of lumbrokinase
Fresh P. praepinguis was cleaned with water several times to remove all the dirty materials on the earth-worms.The surface moisture was absorbed with the filter paper.The weight was taken and then added two volumes of phosphate buffer (pH 7.8).The tissue homogenizer was used to homogenize for 5 min at 4°C.Then the sample was kept standing overnight at 4°C.Finally the sample was centrifuged at 5,000 rpm for 10 min and discarded the pellet.The solid ammonium sulfate was added to the supernatant until saturation.The sample was again centrifuged at 4°C, 40000 rpm for 10 min.The precipitate collected was dissolved in a phosphate buffer solution, dialyzed with a dialysis bag and then desalted using a freeze dryer and freeze-dried to obtain a powdery substance of a large number of light-brown.The crude product was lumbrokinase, stored at 4°C temperature (Astrup and Mullertz, 1952).

Preliminary determination of lumbrokinase activity
The initial activity of lumbrokinase preform was determined by Casein plate method (Astrup and Mullertz, 1952).The size of diameter of the transparent circle represents casein hydrolyzing activity of lumbrokinase.

Determination of the activity of lumbrokinase by casein as substrate
Casein powder (1 g) was added to Tris-HCl buffer (volume: 100 mL, 0.05 mol/L , pH 7.8) and lysed by heating, so that the substrate concentration was 1%.It was incubated at 37°C. 2 mL of lumbrokinase treated enzyme solution was drawn, quickly added 2 mL 37°C pre-insulated substrate solution, shake well, continue to incubate for 30 min, and finally added 2 mL of 10% trichloroacetic acid solution to stop the reaction.The sample was kept at room temperature for 30 min, filtered and then the absorbance of filtrate was determined at a wavelength of 280 nm.

Determination of optimum pH of lumbrokinase activity
Taken lumbrokinase freeze-dried powder into enzyme solution, take 1 mL of enzyme solution were added 2 mL pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 phosphate buffer, mix.After 4 hours at room temperature, the absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated to determine the optimum pH of their lumbrokinase activity.

Determination of optimum temperature and thermal stability of lumbrokinase
2 mL lumbrokinase enzyme solution and 2 mL 1% concentration of casein solution respectively incubated for 10 min at different temperatures including 20, 30, 40, 50, 60, 70, 80, 90°C.Then the enzyme solution mixed with the casein solution at same temperature.Mixed solution was incubated for 30 min at the original temperature.Finally, 2 mL 10% trichloroacetic acid solution was added to the mixed solution to stop the reaction.The mixed solution was filtrated after standing at room temperature for 30 min.The absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated, to determine the optimum temperature.
Two milliliters of enzyme solution was incubated at the optimum temperature conditions for different time including 2, 4, 6, 8, 10, 12, 16 and 24 hours.Then the enzyme solution was mixed with casein solution (1%; 2 mL).The mixed solution was incubated for 30 min at the original temperature.Finally, 2 mL 10% trichloroacetic acid solution was added to the mixed solution to stop the reaction.The mixed solution was filtrated after standing at room temperature for 30 min.The absorbance of filtrate was determined at a wavelength of 280 nm, retention of enzyme activity was calculated, to determine the holding time of lumbrokinase activity under optimum temperature.

Crude extracts and active detection of lumbrokinase
Total 12 g freeze-drying crude product of lumbrokinase was obtained crude extract by ammonium sulfate salting precipitation method with 2053.1 g clean fresh P. praepinguis.Lumbrokinase yield was 0.5% which was relatively low.
Lumbrokinase activity was higher and the average diameter of the transparent circle was 15 mm with casein plate method (Figure 1).

Determination of optimum pH of lumbrokinase activity
Lumbrokinase activity was significantly affected by pH (Figure 2).Lumbrokinase activity was relatively stable Bangladesh J Pharmacol 2016; 11: S106-S109 S107 Figure 1: Lumbrokinase activity detection with casein plate method and high activity in the alkaline environment (pH 7-9), Lumbrokinase activity retention rate maintain 75% when pH increased to 11. Lumbrokinase activity gradually reduced in an acidic environment with pH decreased, lumbrokinase activity retention rate was less than 25% at pH 4 and zero at pH 3.

Effect of temperature on lumbrokinase activity
Lumbrokinase activity was significantly affected by pH (Figure 3).Lumbrokinase activity retention rate was 100% below 60°C, rapid declined less than 10% when temperature raised 70°C and 0% when temperature reached 80°C.
Lumbrokinase activity kept intact and lumbrokinase activity retention rate maintained 100% in temperature range of 20°C to 60°C, but the water bath time of enzyme solution at 60°C also affected the lumbrokinase activity (see Figure 4).Lumbrokinase activity retention rate maintained 100% with water bath 60°C, 2 hours.With the water bath time in 60°C extended, lumbrokinase activity decreased gradually and stabilized with water bath 60°C, 16 hours.Lumbrokinase activity retention rate stabled at around 33.0% after 16 hours of water bath in 60°C.

Discussion
The yield of lumbrokinase crude extract was only 0.525%, lower than 2% in previous studies.Low lumbrokinase yield in this experiment may be related with many factors such as using fresh wet earthworms, not all discharged dirt in earthworms, earthworms homogenized stored at 4°C for long time, and other factors.
Lumbrokinase completely inactivated in pH conditions less than 3, but the stability of lumbrokinase in a neutral or alkaline environment was preferably (Yang and Ru, 1997;Lu et al., 2007).This results maybe related with protein denaturation with strong acid environment.The results in this experiment was completely consistent with the results that the clinical drug of lumbrokinase can be well adapted to alkaline environment in the

Figure
Figure 2: Effect of pH on lumbrokinase activity

Figure 4 :
Figure 4: Effect of water bath time in 60°C on lumbrokinase activity