Fibrin Lab Report

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2.1. Isolation of fibrinolytic enzyme from Paenibacillus sp. IND8
The optimized conditions of fibrinolytic enzyme production by Paenibacillus sp. IND8 were described previously [28]. This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min).
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The incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.…show more content…
Fibrinolytic activity was determined using the fibrin plate method [33]. The fibrin plates composed of 7.5 ml of 1.2% (w/v) fibrinogen (ID BIO, France), 0.05 ml of thrombin (100 NIH units/ml; Biopharm Laboratories, Bluffdale, Utah 84065), 1% (w/v) agarose, and 7.5 ml of 100 mM sodium phosphate buffer (pH 7.4). The plates were left at room temperature (30°C ± 2°C) for 1 h to form a fibrin clot layer. To make plasminogen-free plate, the fibrin plate was heated at 80°C for 30 min to inactivate plasminogen present with the commercially available fibrinogen. Ten microliters of fibrinolytic enzyme was placed on the plasminogen-free and plasminogen-rich plates and incubated at 32°C ± 2°C for 4 h. The enzyme exhibited a clear zone of degradation of fibrin on plasminogen-rich plate and plasminogen-free plate was observed.
2.8. Blood clot lysis of fibrinolytic enzyme
The blood clot lytic effect of fibrinolytic enzyme was studied with an artificial clot in vitro. The blood was collected from a healthy male volunteer with written informed consents. Artificial blood clot was made by spontaneous coagulation in the sterile vials. Then, different doses of the fibrinolytic enzyme from Paenibacillus sp. IND8 were added. Steptokinase was used as the positive control; buffered saline was used as the negative control. These

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