Fig. 5. Semi-in vivo crosslinking identifies four major targets of ovoperoxidase. (A) Isolation scheme for in vitro crosslinking assay, based on the ability to inhibit ovoperoxidase-dependent crosslinking using diphenyleneiodonium (DPI). (B) Titration of tyramide-Alexa Fluor 594 incorporation using the in vitro crosslinking assay from A. Fifty micrograms of DPI-isolated SFEs were reacted with various concentrations of tyramide-Alexa Fluor 594 in the presence of 10 mM tyramine (unless noted) and 10 µM hydrogen peroxide. Two-fifths (20 µg) of each reaction was separated by SDS-PAGE, and then visualized for Alexa Fluor 594 fluorescence (red) prior to staining with Coomassie (blue). Images were overlaid in pseudocolor to identify fluorescently labeled proteins (purple). Coomassie staining identifies the major fertilization envelope bands, including proteoliaisin (PLN), SFE1, SFE9, rendezvin isoforms (RDZ⁶⁰, RDZ⁹⁰) and ovoperoxidase (OVOP). (C) DPI-SFEs were in vitro crosslinked using 10 µM H₂O₂, and then separated by SDS-PAGE. Gels were stained for total protein (5 µg, Coomassie) or immunoblotted for individual fertilization envelope components (1 µg). (D) Titration of in vitro fertilization envelope crosslinking of rendezvin. Five micrograms of DPI-SFEs were exposed to serial dilutions of hydrogen peroxide (maximum of 10 µM H₂O₂) in the presence or absence of the inhibitors 3-AT (1-100 mM) or DPI (10 µM). One-fifth (1 µg) of each reaction was separated by SDS-PAGE and either stained with Coomassie or immunoblotted for rendezvin (RDZ). Individual Coomassie bands of structural fertilization envelope proteins are labeled. Arrow indicates rendezvin¹²⁰.