I want to determine Glucokinase activity of the crude enzymes from Streptococcus pyogenes. I found the following method from the internet.
Method: http://www.worthington-biochem.com/HK/assay.html
The assay is based upon the reduction of NAD+ through a coupled reaction with glucose-6-phosphate dehydrogenase and is determined spectrophotometrically by measuring the increase in absorbance at 340 nm.
Instead of using NAD can I use NADP in this assay?
The title of you question and the text are two different things. I take it the title should be .
I would think it much safer to stick with NAD+, and I would have thought it to be (a) available and (b) cheaper. Gthere is work by David Berkowitz (Nebraska) on this topic. Look it up.
GMB
There is a method using the NADP+, see the paper, Gibon et al 2004 (plant Cell). This method is a stop assay, first part using a coupled reaction with the glucose-6-phopshate dehydrogenase and second part using a cycling system....
GM
for Glc6PdH either NAD+ or NADP+
http://www.worthington-biochem.com/zf/default.html
but
look @ Specificity
http://www.worthington-biochem.com/zf/cat.html
Greetings Marko
I think it is much better to use NADP+ and high glucose concentration (20 mM or more)
You can use either. What you need is the acceptable hydrogen acceptor to oxidize glucose 6 phosphate. NAD+ or NADP+ is acceptable
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