Fig. 4.

Quantitative hemolysis assays with rabbit erythrocytes. (A) One-hour activity assays on HlyII and αHL synthesized by IVTT. The first well in each panel contained IVTT mix diluted to a final volume of 100 μL in MBSA. The concentration of αHL in an IVTT mix was determined as previously described (Walker et al. 1992b; Miles et al. 2001) and used to calculate the concentration of HlyII produced by IVTT (see Materials and Methods). On this basis, the final concentrations of HlyII or αHL were made equal in the first well (0.21 μg/mL). Twofold serial dilutions from left to right were then made. The final concentration of rRBC in all wells was 0.5%. The arrows indicate ∼50% lysis. (B) One-hour activity assays on various constructs synthesized by IVTT. IVTT mixes containing HlyII, HlyII(ΔCT), αHL, and αHL-TL were diluted 10-fold with MBSA before addition (1 μL) to the first well. TL, the C-terminal extension of HlyII was translated as a separate polypeptide, and added where indicated in 32-molar excess as determined by phosphorimager quantitation (5 μL of undiluted IVTT mix in the first well). Twofold serial dilutions were made from left to right.