FIGURE 4.

Alkylation of the Cys⁴⁶² and Cys⁵⁴¹ plasmin thiols inhibits PGK-mediated angiostatin formation. A, plasminogen (20 μg) was untreated or alkylated with 20 mm iodoacetamide (IAM) for 24 h. The plasminogen was converted to plasmin, and 20 μg was incubated with 80 μg of PGK in 0.5 ml of Hepes-buffered saline for 30 min at 37 °C. Plasmin products were separated on SDS-PAGE and blotted with polyclonal anti-plasminogen antibodies. The angiostatin fragments are indicated. The positions of molecular mass markers are shown at left. B, plasminogen (6 μg) was incubated with 50 mm iodoacetamide for 8 h and then digested with chymotrypsin. Tandem mass spectrum of the chymotryptic fragment, LPNVETPSEEDCMF, shows Cys⁴⁶² (underlined) labeled with the iodoacetamide adduct, carboxyamidomethyl. The accurate mass spectrum of the peptide is shown in the inset (observed [M+2H]²⁺ = 834.3497 m/z; expected [M+3H]³⁺ = 834.35 m/z).