1) NH3 is generated from the substrate
2) NH3 + NADH + α-Ketoglutarate -> NAD+ + Glu
In the reaction, samples with a higher substrate concentration should produce more ammonia and consume a higher amount of NADH for glutamate generation, and thereby showing the greatest decrease in absorbance at 340nm (A340) as the reaction progresses.
Using a fixed starting concentration of NADH, the initial A340 is supposed to be the same across different samples. However, the blank which should not have any NADH consumption at all has the lowest A340.
At the final time point, trend can be generally observed that A340 decreases with increasing substrate concentration but this trend is lost in lower substrate concentration.
Is there any explanation on the weird results?
Other things in your sample besides NADH may have an absorption at 340 nm, causing the blank to have the lowest initial A340. However, this is not necessarily a problem if you base your activity measurements on the slope of the A340 nm decrease.
To maximize the rate of consumption of ammonia by the glutamate dehydrogenase reaction, make sure the concentrations of α-ketoglutarate and NADH are saturating. Make sure the coupling enzyme concentration is not rate-limiting by testing the effect of increasing its concentration on the rate of NADH consumption.
It may help to optimize the buffer conditions for the coupling enzyme, rather than the primary enzyme.
Consider where the equilibrium for the 2nd reaction lies. If it is not strongly to the right, then you may have to add a second coupling enzyme to consume the product of the first (something that use glutamate as a substrate) to keep it going.
To me it sounds like NADH is consumed in your blank reaction. Can ammonia be released from your substrate? Or may your enzyme consume NADH? (I guess you would know that)
Considering your last point - what is the origin of your substrate? Is it commercial or home-produced? Can it contain ammonia? One of our students had similar problem currently.
Anyway, as Adam pointed out, as long as you include the blank reaction, it's OK. I had worked for years with "enzyme", which had very high background activity.
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