Fig. 4. Identification of ovoperoxidase target proteins labeled in vivo. (A) Fertilization envelopes isolated from embryos fertilized in the presence of 5 mM tyramine alone, or with tyramide-Alexa Fluor 594 or tyramide-biotin (1:100 dilution each) were separated by SDS-PAGE. Sixty micrograms of purified fertilization envelopes were used for Coomassie staining, the identification of Alexa Fluor 594 fluorescence, and detection of biotinylated bands. Antisera for rendezvin (RDZ) epitopes or for ovoperoxidase (OVOP) were used to probe 2 µg purified fertilization envelope proteins by immunoblot to identify the major target of ovoperoxidase-dependent dityrosine crosslinking (arrowhead). The major fluorescently labeled protein is boxed. (B) List of unmodified peptide matches to rendezvin¹²⁰ from mass spectrometry analysis of fluorescently labeled band from the fertilization envelopes (boxed band from A). (C) Fifty micrograms of total zygote lysates fertilized in the presence or absence of 3-AT or the Udx1 inhibitor diphenyleneiodonium (DPI), which abolishes synthesis of the ovoperoxidase substrate hydrogen peroxide (Wong et al., 2004), were separated by SDS-PAGE, and then stained with Coomassie or immunoblotted for different rendezvin (RDZ) epitopes. RDZ anti- is against the amino-terminal fragment whereas RDZ anti- is against the carboxy-terminal portion.