A real-time observation of HK-Mac-1 interaction.

(A) Overlay sensor-gram from the IAsys showing the binding of Mac-1 to immobilized HKa. At “1” purified human Mac-1 was added to the cuvette at the following concentrations: 5, 10, 20, 40, 60, 80, 120, and 160 nmol/L. At “2” the cuvette was washed with buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 50 μmol/L ZnCl₂, and 0.05% Tween-20). (B) dR/dt plots of association data according to equation 3. Straight lines were fitted to the initial linear portion of the association phase data to obtain k_s. (C) Plot of k_s versus Mac-1 concentration. The slope of the line gives the association rate constant. (D) Dissociation phase data from 160 nmol/L Mac-1 sensor-gram plotted according to equation 5. A straight line was fitted to the first 20 seconds of the plot for phase 1, and the other straight line was fitted to the data between 20 and 70 seconds for phase 2.