Fig. 6

Zebrafish Alr is a flavin-linked sulfhydryl oxidase.

A. Alr is present as dimers. Recombinant zebrafish Alr purified from E.coli was examined by SDS-PAGE and stained by commassie blue. In the presence of the reducing reagent DTT, Alr protein is in the monomer form, with a size of around 23 kD. In the absence of DTT, both Alr and the mutant Alr^C131S are present as dimers with sizes in the 40~46 kD region. B. Absorption spectra of recombinant zebrafish Alr and Alr^C131S protein at 15 µM. Free FAD at 15 µM was used as reference and its spectra show the typical riboflavin spectrum, with two absorbance peaks at around 375 nm and 450 nm. The absorption spectra of both the wild type Alr protein and mutant Alr^C131S protein are characteristic of the FAD moiety, with a minor shift of the first peak to 365 nm compared to the free FAD. Under equal molar concentration, the amount of Alr-bound FAD is only half of the free FAD, indicating about 50% loading of FAD in the recombinant Alr preparation. C. Alr is a sulfhydryl oxidase. Enzymatic assay using DTT as substrate, showing the reduction of free thiol groups overtime. The blue line represents DTT alone. Wild type Alr protein oxidized thiol groups over time while the CxxC motif mutant, Alr^C131S, completely lost the sulfhydryl oxidase activity.