Ing buffer, for one hour at ambient temperature.
Following washes with
Ing buffer, for one hour at ambient temperature. Following washes with PBS-T, colour was developed using a freshly made solution of 5 mg o-phenylenediamine (Pierce) in 12 mL of 25 mM citric acid, 97 mM Na2HPO4 pH5.0 supplemented with 0.03 (v/vol) hydrogen peroxide for ten minutes prior to stopping the reaction with sulfuric acid. The optical density at 490 nm was then read on an EL808 Plate reader (Biotek Instruments, Winooski, VT). In some experiments, competitor peptides were added with the thrombin; in others S195A-thrombin or thrombin inactivated at its active site using D-Phe-L-Pro-L-Arg chloromethylketone (FPR-ck, EMD Millipore, Billerica, MA) was substituted for -thrombin. Binding isotherms such as those shown in Figure 1A were curve-fit for one-site binding (hyperbola) using non-linear regression in GraphPad Prism 4.0 software (GraphPad Software, La Jolla, CA). The same program wasBoyle et al. BMC Biochemistry 2013, 14:6 http://www.biomedcentral.com/1471-2091/14/Page 4 ofsodium diethyl barbiturate, 4-d][1 131 mM NaCl pH 7.4) containing or lacking HCII- or hirudin-related peptides, and clotting time was determined following the addition of 100 L Thrombin 10 reagent.Biotinylation of peptides and proteinsHCII- or hirudin-related peptides in 50 mM sodium phosphate pH 6.5 were combined with 20-fold molar excess sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHSLC-biotin, Pierce) at ambient temperature for 30 minutes. Reactions were quenched with excess Tris-Cl pH 7.4 and sulfo-NHS-LC-biotin removed by desalting or dialysis. Biotinylated FPR chloromethylketone (bFPRck; Haematologic Technologies, Essex Junction, VT) was used to modify -thrombin at a bFPRck: thrombin molar ratio of 10:1 in Tris-buffered saline (TBS, pH 7.4) for ten minutes at ambient temperature. Excess reagent was removed by overnight dialysis versus TBS. Thrombin biotinylated 3-Bromo-4-(propan-2-yloxy)aniline hydrochloride by bFPRck had no detectable amidolytic activity against S2238.Surface plasmon resonanceFigure 1 Binding tert-Butyl (2-bromothiazol-5-yl)carbamate of different forms of thrombin to HCII 1-75 immobilized on microtiter plate wells. Binding of -thrombin (-IIa, solid squares) or T-thrombin (T-IIa, ) was measured as the optical density (OD) at 490 nm as described in Materials and Methods, and graphed relative to the mean OD of binding reactions carried out with 100 nM -thrombin, taken as 100 , in Panel A. The mean ?the SD of 3 independent experiments is shown. Panel B shows the results of experiments similar to those shown in Panel A, but carried out with 12.5 nM -thrombin (-IIa), S195A-thrombin (S195A-IIa), FPR-ck -thrombin (FPR-ck-IIa) or T-thrombin (T-IIa); results were normalized to the mean optical density (OD) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17591728 at 490 nm observed for -thrombin binding. Each bar represents the mean ?the SD of 3 independent experiments; * indicates p<0.05 by non-parametric ANOVA with Dunn's post-test versus -thrombin binding.used to calculate IC50s in competitive binding experiments in which HCII-derived or hirudin-derived peptides were used to compete the binding of thrombin to immobilized HCII 1-75. Inhibition plots such as those shown in Figure 2 were PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 logarithmically transformed, lines of best fit solved by linear regression, and the competitor concentration reducing binding to 50 (IC50) was determined.Thrombin clotting timesSurface plasmon resonance experiments were carried out using a BIAcore X biosensor instrument (Biacore, Piscataway, NJ) at 25 . Biotinylated HCII- or hirudinrelated peptides were immobilized on streptavidin-coated sensor chips (GE Hea.
