FIGURE 2.

Substitutions in the upper hinge and related impacts to radical induced hinge cleavage. Nine mutants were designed to determine any roles of Asp²²⁵ and His²²⁹ in the hinge cleavage. These substitutions were constructed by introducing single or double substitutions in the upper hinge of the native sequence by site-directed mutagenesis. Substituted residues are underlined (panel A). These mutants were evaluated for their ability to inhibit the hinge cleavage by SEC to measure the degraded products (Fab and a partial IgG1), the SEC was performed after incubating the IgG1 with H₂O₂ in a molar ratio of 1:200 at 25 °C for 2, 4, and 6 days, respectively. The cleavage products (% LMW) are shown as % of the peak area of the products to the whole molecule in the UV signals (panel B). For clarity, only the results from Day 6 and Day 0 are shown here. Percent change was calculated by comparing the level of % LMW at Day 6 to Day 0. Percent inhibition was calculated by the equation: % = (% change of the native − % change of mutant)/% change of the native. LMW: low molecular weight species (Fab and a partial IgG1). The results are reported as an average of two replicates.