Apparently catalase can bind NO, but every other publication I've seen where this protocol has been used (usually as a negative control for endothelial dysfunction studies) shows negative results, yet mine were very conclusive
Catalase reacts with NO and produce ferricatalase-NO- not active enzyme form.
Superoxide (O2-) could converted the inhibited ferricatalase-NO to the active ferricatalase. Such reactivation could be observed in system with simultaneous generation of NO and O2-, so if your NO donor is something like 3-morpholinosydnonimine (in fact peroxynitrite donor) . NO can be destroyed fast in such condition. Did you try several different NO donors with different NO release mechanism?
Karel, thank you for your answer. I used MAHMA NONOate because liberates NO in buffer. I avoided using SNP because I believe this releases O2- when bioactivated intracellularly. Your answer makes perfect sense, but I thought it odd that no one else seemed to find this. All published data I have seen shows that catalase does not affect vascular relaxation to SNP or NONOates, whether studied in healthy or diseased tissue. Essentially this type of experiment is used simply as a negative control to demonstrate specific breakdown of H2O2.
Do you add endogenous catalase in your organ during bath vasorelaxation experiments? I was once testing different protein than catalase and I realized that it is decomposed quickly. I have oxygenated organ bath and there was a foam on the top when protein was added and probably it caused its denaturization then you have free Fe-Hem in the system....I will try mix your NO donor with catalase and test if your NO release is changed. Catalase should produce oxygen maybe this could oxidase NO to nitrite / nitrate and NO could not reach GC. Anyway interesting observation make all proper control (dose depend curve etc) and publish it.
Yes it was oxygenated (95% O2, 5% CO2) Holmans buffer at 37C, and catalase was added at final bath concentrations of 500, 1000 and 2000 Uml-1. Exogenous catalase did foam when added to the bath (in all experiments), but apparently this is "normal". It was a while ago that I did these experiments (2009ish, during my PhD studies). You can download my PhD thesis from Cardiff University website http://orca.cf.ac.uk/55885/, the results are in the Appendix at the back.
I did consider using my colleague's chemiluminescence system to see if NO was consumed by catalase but I was at the end of my studies and simply didn't have the time
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Medical Pharmacology
- Clinical Pharmacology