converting enzyme activity units

[northernlad2690]

Hello, I'm just wanting someone to give me a little bit of guidance on the following conversion,

i'm trying to convert my reading of umol/min/umol to umol/min/mg- i think i have the correct answer but i need my method validated if someone could possibly help?

i have a spectrophotometric assay of a purified recombinant enzyme, Spy057, against p-nitrophenyl N-acetyl-β-glucosaminide substrate (total reaction volume, 2 mL; volume of Spy05783 added, 50 µL):

- I have calculated the umol/min/umol using beer lamberts law to 289.63 

- the Mr of spy057 is 43507g/mol

- i then calculated that there is 43.507mg/umol

- there is 1.899 x 10E-4 umol added reaction volume 

so using this i then calculated 43.507x(1.899E-4) = 8.272 x 10E-3 

using data obtained previously i knew that the umol/min of p-nitrophenol was 0.055 so this was input into the final equation 0.055/8.272 x 10E-3 giving me 6.657umol/min/mg

does this look correct, ive done everything to try and find a protocol but cant find anything

[studiot]

Forgive me but what's a micromole per minute per micromole?

 

I've never heard of them.

[northernlad2690]

I'm using it for the calculation of an enzyme assay when calculating the enzyme activity of a recombinant protein.

It indicates umol of substrate catalysed per minute, per umol of recombinant enzyme added to the assay. 

- first time I've ever used this specific methods for calculating enzyme activity but is being used prior to calculating katal.

[BabcockHall]

I only have time for a quick comment right now.  I have coached many people through the process of calculation of rates.  A common error is to use the wrong volume when converting from absorbance per unit time into µmoles per minute.  But if you are confident that you have done that correctly, then great.