an odd-appearing Michaelis-Menten curve

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jak3099
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an odd-appearing Michaelis-Menten curve

Post by jak3099 » Fri Jul 27, 2007 1:07 am

Hi all,

I have been assaying an enzyme that oxidizes OH groups and reduces NADP+. The assay is based on the absorbance of NADPH at 340 nm.

The problem is that the graph of reaction rate versus substrate concentration looks like an exponential curve rather than the usual Michaelis-Menten curve I have obtained for other mutants.

Has anyone encountered this problem? I've tried using freshly purified enzyme, changing the amount of enzyme and NADP+.

Thanks in advance! :)

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victor
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Post by victor » Fri Jul 27, 2007 4:05 am

I think that Michaelis-Menten curve is shaped like exponential curve. And what do you mean by a usual curve?
um, would you show the curve here? :?
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blcr11
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Post by blcr11 » Fri Jul 27, 2007 10:35 am

By "exponential" I take it you mean it never saturates. Try lowering the amount of enzyme. You may have too much and you just haven't increased the substrate concentration enough to reach saturation. Another possibility is that you've got some kind of funny cooperativity going on. (The standard Michaelis-Menton curve is sigmoidal-at least it is prior to doing things like Lineweaver-Burking or Eadie-Hofsteadering.)

jak3099
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Post by jak3099 » Sat Aug 04, 2007 12:10 am

thanks, i'll try lowering the enzyme amount/increasing substrate concentration. :D

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