Binding between purified XD and purified N TAIL proteins was analyzed by using BIAcore 3000 (Amersham Pharmacia Biotech), a system for real-time biomolecular interaction analysis that is based upon surface plasmon resonance technology. Purified XD (1.4 mg/mL in acetate buffer at pH 5.5) was covalently bound to carboxy-methyl groups of CM5 sensor chips using amine-coupling chemistry (Biosensor AB, Amersham Pharmacia Biotech). The levels of immobilized XD were comprised between 180 and 225 RU (1000 RU equal a change in mass of 1 ng/mm 2 on the sensor surface) (Zhang et al. 2002) . Kinetic and equilibrium constants were calculated from global analysis of reactions with multiple analyte concentrations (0.1, 0.2, 0.5, 1, 2, 5, and 10 mM). Reactions were performed at 25 C. Remaining flow channels on the sensor chip included a control for a nonspecific interaction with the sensor chip (i.e., activated/ blocked flow channel) and a control for nonspecific interactions with irrelevant protein targets (i.e., lactoferrin conjugated flow channel). Protein analytes were passed over the sensor chip in HBS-P buffer (0.01 M HEPES at pH 7.4, 0.15 M NaCl, 0.005% surfactant P-20) containing 2.5 mM magnesium acetate and 2.5 mM ATP. Sensorgrams plotted changes in surface plasmon resonance (measured in RU) as a function of time. Multiple sensorgrams representing various analyte concentrations were analyzed by using BIAevaluation 3.1 software. Background interaction of N TAIL proteins (i.e., analytes) with the sensor surfaces were measured on flow channels that were activated and subsequently blocked under buffer conditions used to immobilize XD. This background was subtracted from all binding curves (i.e., sensorgrams) prior to global analyses. Immobilized lactoferrin was used as a specificity control, and the resultant sensorgrams ruled out high affinity interactions between N TAIL constructs or peptides and this irrelevant protein ligand (data not shown). Global fitting of experimental data to well-characterized binding reactions was used to define reaction rate and equilibrium constants. Signal changes on the activated/ blocked control channel were subtracted from the peptide-XD and N TAIL protein-XD interactions using in-line reference and the subtracted sensorgrams were analyzed. Curves generated with serial analyte concentrations were applied globally to the 1:1 Langmuir binding model with or without correction for baseline drifting depending on baseline status. 2 and residual values were used to evaluate the quality of fit between experimental data and individual binding models. Plots of residuals indicate the difference between the experimental and reference data for each point in the fit. The 2 value represents the sum of squared differences between the experimental data and reference data at each point. A good fit between experimental and reference data has small residuals in the À2 to +2 range that randomly distribute about the X-axis, and 2 values are less than 10.