Illinois State University, Chemistry, Normal, IL 61790
The effects of covalent cross-linkers on the enzyme, coproporphyrinogen oxidase, had been previously studied but their role in protecting the enzyme from protease cleavage has not been evaluated. Therefore, we examined how the protein cross-linker BS3 affects the ability of trypsin to digest purified, wild-type recombinant human coproporphyrinogen oxidase and selected mutants. Following incubation, the apparent molecular weights were evaluated by SDS-PAGE and enzymatic activity was monitored by spectroscopy following HPLC. For both wild-type and mutants, the results indicated that the cross-linker was indeed able to protect against trypsin digestion relative to the enzyme incubated with trypsin in the absence of the cross-linker. In fact, one of the mutant enzymes was rendered completely nonfunctional when trypsin addition was followed by BS3 addition. All of these data have implications for the episodic nature of porphyria, a rare, inherited blood disorder.
[Abstract (DOC)]