Variation of active single-chain fragment, variable (scFv) concentrations in periplasmic extracts with time at 25°C. (A) ln(% binding rate at time 0) as a function of time. The scFv1F4-WT (diamonds) and mutants RL24S (circles) and RL24S/DL70T (squares) were used at two initial concentrations of 57 and 101, 43 and 95, and 57 and 97 nM, respectively. Mutant DL70T (triangles) was used at an initial concentration of 30 nM. (B) Histogram representation of the TA50 values calculated for scFv1F4-WT and the three mutants of the H46-H62 pair, in two independent experiments.