0.4 mM BApNA (Sigma) for trypsin and 0.4 mM Suc-AAPF-pNA (Sigma) for a-chymotrypsin. Additionally, 400 ng of human plasmin (Sigma), thrombin (Sigma), tissue plasminogen activator (tPA; Sigma), or factor Xa (Novagen) was incubated with increasing amounts of Bi-KTI or aprotinin (Sigma) at 37uC for 30 min in 50 mM Tris-HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 0.5 mM of chromogenic substrate (Chromogenix, Mo¨ lndal, Sweden): S-2251 for plasmin, S-2238 for thrombin, S-2288 for tPA, and S-2222 for factor Xa. Fibrinolytic cleavage assay Human fibrinogen (200 mg, Sigma) that had been clotted with 1 unit of thrombin in 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM CaCl2 was incubated with plasmin (500 ng) or both plasmin and Bi-KTI (16.6 ng) at 37uC. The fibrinolytic cleavage was analyzed using 12% SDS-PAGE (10 mg/lane). Fibrin plate assay The fibrin plate assay was performed with 5 ml of human fibrinogen (0.5%) clotted with three units of thrombin. Plasmin or a mixture of plasmin and Bi-KTI was dropped onto the fibrin plates, and the plates were incubated at 37uC for various periods of time. The fibrinolytic activity was determined by examining the formation of a clear area [13]. Plasmin inhibitory assay Human plasmin (25 nM, Sigma) was incubated with increasing amounts of Bi-KTI or aprotinin (Sigma) at 37uC for 30 min in 50 mM Tris-HCl buffer (pH 7.4), and the residual enzyme activity was determined at 405 nm using 100 mM S-2251. The initial reaction rate was determined by calculating the slope of the linear portion of the kinetic curve. The inhibitory effect was expressed as the percent reduction in the initial hydrolysis rate; the reaction rate in the absence of inhibitor was taken as 100%. The inhibitor References 1. Neurath H (1984) Evolution of proteolytic enzymes. Science 224: 350–357. 2. Krem MM, Cera ED (2002) Evolution of enzyme cascades from embryonic development to blood coagulation. 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(2010b) Dual strategy of bee venom serine protease: prophenoloxidase-activating factor in arthropods and fibrin(ogen)olytic enzyme in mammals. PLoS ONE 5: e10393. Bee Venom Serine Protease Inhibitor concentration that decreased the rate of hydrolysis by 50% (IC50) was determined. The values of the inhibition constants (Ki) were calculated using the equation Ki = IC50/(1+S/Km) [25]. Electrophoretic mobility shift assay Plasmin (three mg) in 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM CaCl2 was mixed with 1 mg of Bi-KTI and incubated at 37uC for 1 h. Samples were resolved on a 10% polyacrylamide gel at 4uC. Following electrophoresis, the proteins were blotted onto a sheet of nitrocellulose transfer membrane (Schleicher & Schuell, Dassel, Germany), and the membrane was blocked by incubation in a 1% bovine serum albumin (BSA) solution and then incubated with an antiserum solution (1:1,000 v/v) against plasmin or the His-tag at room temperature for 1 h. 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PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32269