A new inhibitive heavy metals determination method using trypsin has been developed. The enzyme was assayed using the casein-Coomassie-dye-binding method. In the absence of inhibitors, casein was hydrolysed to completion and the Coomassie-dye was unable to stain the protein and the solution became brown. In the presence of metals, the hydrolysis of casein was inhibited and the solution remained blue. The bioassay was able to detect zinc and mercury with IC50 (concentration causing 50% inhibition) values of 5.78 and 16.38 mg l(-1) respectively. The limits of detection (LOD), for zinc and mercury were 0.06 mg l(-1) (0.05-0.07, 95% confidence interval) and 1.06 mg l(-1) (1.017-1.102, 95% confidence interval), respectively. The limits of quantitation (LOQ) for zinc and mercury were 0.61 mg l(-1) (0.51-0.74 at a 95% confidence interval) and 1.35 mg l(-1) (1.29-1.40 at a 95% confidence interval), respectively. The IC50 value for zinc was much higher than the IC50 values for papain and Rainbow trout, but was within the range of Daphnia magna and Microtox. The IC50 value for zinc was only lower than those for immobilized urease. Other toxic heavy metals, such as lead, silver arsenic, copper and cadmium, did not inhibit the enzyme at 20 mg l(-1). Using this assay we managed to detect elevated zinc concentrations in several environmental samples. Pesticides, such as carbaryl, flucythrinate, metolachlor glyphosate, diuron, diazinon, endosulfan sulphate, atrazine, coumaphos, imidacloprid, dicamba and paraquat, showed no effect on the activity of trypsin relative to control (One-way ANOVA, F(12,26)= 0.3527, p> 0.05). Of the 17 xenobiotics tested, only (sodium dodecyl sulphate) SDS gave positive interference with 150% activity higher than that of the control at 0.25% (v/v).
The core protein (HBcAg) of hepatitis B virus (HBV) has been shown to interact with the large surface antigen during HBV morphogenesis, and these interactions can be blocked by small peptides selected from either linear or constrained phage display peptide libraries. The association of HBcAg with peptide inhibitors was quantitatively evaluated by isothermal titration calorimetry. The thermodynamic data show that the interaction between HBcAg and peptide MHRSLLGRMKGA is enthalpy-driven and occurs at a 3:1 stoichiometry and dissociation constant (Kd) value of 79.4 muM. However, peptide WSFFSNI displays a higher binding affinity for HBcAg with a Kd value of 18.5 muM when compared to peptide MHRSLLGRMKGA. A combinatorial approach using chemical cross-linking and surface-enhanced laser desorption/ionization-time-of-flight-mass spectrometry shows that the Lys of peptide MHRSLLGRMKGA interacted either with D64, E77, or D78 of HBcAg.