Figure 5.

Dependence of protein splicing and intein reassociation on pH. Experiment 1 (• and ○): Mixtures of MU_NΔ and U_CΔH were reconstituted under standard conditions at pH 7.5 and diluted 2-fold into 200 mM buffers at the pH values indicated, by using either phosphate buffers (•) or Tris buffers (○). Protein splicing was initiated by the addition of DTT to 1.25 mM, was allowed to proceed at 25°C for 6 hr, and was assayed by Western blot analysis with anti-His-tag antibody. The relative amounts of MH were estimated by densitometry of the Western blot. Experiment 2 (▪ and □): Mixtures of MU_NΔ and U_CΔH were reconstituted at the pH values indicated, by using either phosphate (▪) or Tris buffers (□). Protein splicing was allowed to proceed under standard conditions for 6 hr and was assayed as in experiment 1. The densitometry values for reassociation were normalized to those for splicing by using the point for Tris⋅HCl, pH 7.5, as a common reference point.