Titration of a fluorescein labeled CD4 tail peptide with HIV Nef-His proteins. 1.0 μM of the CD4 peptide in PBS was incubated for 30 min with Nef-His proteins at the concentrations from 0.01 to 10 μM in 0.5 ml volume. Fluorescence emission was recorded with a FluoroMax-2 fluorescence spectrometer (excitation at 492 nm; emission at 516 nm) in a stirred cuvatte at 25°C. Reduction in fluorescence emission after incubation with a protein is expressed as the percentage of the fluorescence before incubation. The reduction in fluorescence is plotted against Nef-His concentration. The values are the average of three repeats. Left panel: Fluorescence reduction of the CD4 peptide after incubation with unalkylated Nef (wt)-His (black circle) or TPCK-alkylated Nef (wt)-His (white square). Right panel: Fluorescence reduction of the CD4 peptide incubated with the untreated Nef (C55A)-His (black circle) or TPCK-treated Nef (C55A)-His (white square).