One-pot
gelation
in capillary glass tubes with carbonate-based
buffer solution allows the formation of hollow collagen gels (collagen
tubes) with an outer diameter of 1 mm or less. The preparation conditions
of collagen concentration, buffer concentration, and capillary diameter
impacted the ratio and size of the hollow gel and allowed for morphological
control of the cavity. The morphology of the hollows suggests that
their vacancies are the result of macroscopic phase separation and
pinning due to gelation. Mechanical strength measurements of the dried
collagen gel tubes demonstrated that the collagen concentration determines
their Young’s modulus and maximum stress and that the material
is strong enough for practical use. In vitro seeding studies of vascular
endothelial cells demonstrated the possible formation of endothelial
cells in layers in the gel lumen.
Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.