help with enzyme charactization PLEASE

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borntoclone
borntoclone's picture
help with enzyme charactization PLEASE

I have a question about a project I am working on, I am new to the biochemistry so any help with be highly appreciated......
I have an enzyme which is supposed to be a lysyl oxidase I say suppose because I have to find it's charactizations ex. pH optimum, temp. ect ect. I also did a kinetic run on my enzymes (two of them) and determined with The Amplex Ultra red and horseradish peroxide assay that these enzymes do produce Hydrogen peroxide. So if this is the case that they are lysyl oxidases then they should also produce ammonium which I also did a filterpaper (for color development)assay on with ammonium sulfate (in different concentration)as a standard. However the one enzyme that in my kinetic study that did not produce a high RFU number for hydrogen peroxide did gave a stong color on the filter paper assay and the enzyme that did produce a high RFU reading for hydrogen peroxide gave little to no color on my filter assay for ammonium detection. Now my question is what can the cause of this be? Besides the fact that the one enzyme that did not produce any ammonium color on the filter may not really be a lysyl oxidase. Could it have something to do with the concentrtion of the enzymes? And wouldn't that be unlikely since you would assume that the enzyme that gave a low RFU reading should be of less concentration?...can someone help me with what to do next?

R Bishop
R Bishop's picture
borntoclone,

borntoclone,

Have you fixed your problem yet? If not did you first determine the concentration of your enzymes or are they in a rough lysate?

Do all lysyl peroxidases produce ammominum?

finally if you arent at the pH optimum would your enzyme work correctly?
could that explain your differential activities?

my initial thoughts

Rb