With regard to your 340-nm increase in absorbance, even in the absence of substrate: make sure that your protein (or something else in the assay solution) is not precipitating over time, because that would give you turbidity and an increased readout at the spectrophotometer. One quick way to check for that is measuring your kinetics in the absence of NAD+.

Hi, as NAD is a bit acidifying in solution this may not be stable and buffered anymore after a while showing persistent decreasing in absorbance 

Hello, you should check in other pH condition. I recommend phosphate buffer pH 7,0 50mmol.  I'm  not sure if I understand but. at 340 nm NAD+ does not have a good absorbance. NADH is the one which has absorbance at 340... For your essay is always important considerate a blank without enzyme and a blank without the substrate, because this cofactor is very unstable. 

All of best

Hi there,

NAD+ stock solution has to be prepared at neutral pH so as it is most of the time sold as a chlorhydrate powder you should neutralize it with NaOH for instance. I have already observed this phenomenon when diluting NAD+ in pH9 reaction mix. I don't know what it is due to but in my case the absorbance signal stabilized within minutes (probably due to the pH transition). So I would suggest you to do the same and wait a bit longer before adding the missing reagent for NADH formation.

Hi Dominique,

You are right,  my enzyme assay has to be at alkaline ph9 which is the optimal pH for my protein. As you said, the signal tend to stabilize after a few minutes.  I will add other reagents until the absorbance  stabilize. 

I read that NAD/NADH degrade at acidic or alkaline condition, I prepare my NAD+ stock in neutral water and did do any neutralization assuming that it should be near neutral and stable for a while. Its weird to see 340nm  absorbance decreases with NAD coz only NADH should absorb at this wavelength.

The signal you get at 340nm depends on the concentration of NAD+. If it's millimolar, you will see something when adding NAD+ (I have estimated the epsilon around 50 M-1.cm-1 at 340nm for NAD+). This signal is actually due to the width of the specific absorption peek at 260nm (if millimolar the heigth of the peek at 260nm will be 18, and as the width of the peek is proportional to its heigth, there will be some absorbance noticable at 340nm even if it's not a specific absorption process).

About NAD+ preparation, make sure you neutralize the HCl associated to NAD+ if powder is a chlorhydrate (if preparing 10mM stock solution of NAD+, add 10mM of NaOH and mix thoroughly).

I noticed that a concentrated (100 mM) solution of NAD turned a bit yellow when I let the pH go alkaline by accidently adding too much base so that the pH was between 9 and 10. I don't know what causes this color. NAD should be colorless. So the 340 nm decrease you see may be due to some chemical change at alkaline pH.

Hi Dominique,

I made my NAD stock to 60mM, the NAD I used is the NAD crystalline hydrate form which doesn't contain chlorhydrate, so should be alright. Thanks!

If your enzyme has a optimum pH at pH 9, it does not mean that you have to measured at that pH. You better use a buffer at physiological pH (less alkaline).

Also sometimes if the reagelt is old it can be degradate by contamination (microbial).

If the change in absorbance in your control  is not considerable according to absorbance change by enzyme activity, you can measured in that conditions.

Hector Nolasco

Thanks Hector! You are right. Theoretically, the Km shouldn't change if we use more or less protein or test under different pH, right?  The signal tends to stabilize within 5mins and is considered not significant to my reaction in the end. I believe that even at alkaline condition such as pH9, the recognized NAD/NADH degradation is considered a slow process, so shouldn't be a serious problem since enzyme assay is measured in a short time!  

Be careful Yong, Km might be dependent on pH if binding / catalytic processes are dependent on protonation/deprotonation state of substrate / aa side chains in the active site ...

Got it, thanks Dominique!

Hi,

I got another weird observation with my enzyme assay. The control reaction containing just NAD+ and my enzyme sample without the addition of substrate displays consistent signal increase at 340nm. I am testing a polyol dehydrogenase, before I suspect that it may be due to the glycerol I added to for long term enzyme storage. However, this observation persists after I freshly purify the protein again without the use of glycerol.

Anyone has any idea how this could happen? Could it be DTT reducing NAD+? The purification buffer I used is 100mM Tris-HCl, 0.5M NaCl, 1mM DTT and imidazle for 6his purification. Why could the 340nm absorbance increase without the addition of any substrate. This is only a problem with some of my proteins  I am working on, while others are fine. Help please! 

Thanks!

One thing you do to investigate is take the UV absorbance spectrum before and after adding the enzyme to see if the 340 nm absorbance increase is due to reduction of NAD to NADH (absorbance peak after reaction is at 340 nm) or something else.

DTT by itself will not reduce NAD to NADH, but maybe your enzyme can use DTT as an electron donor for reduction of NAD. Is the concentration of DTT in the reaction sufficient to account for the absorbance change if this happens?

Hi 

I did a control reaction with the purification fraction that doesn't contain my target protein but contain my purification buffer, the 340nm signal won't change any more, I wonder the protein produced in E.coli LB bound something that could be oxidexed by the enzyme, could this happen?

The enzyme could have an electron donor bound to it when purified, but if this is the source for NAD reduction, you would see only a single turnover of reduction. So if, for example, you had 100 nM enzyme in the reaction, with one mole of electron donor bound per mole of enzyme, then you would see 100 nM of NAD reduced. This would be undetectable by A340.

Try running the reaction without DTT.

Hi Adam, thanks for the suggestions! I will do a control test by eliminating the DTT to see how it goes. It is very hard to estimate how many enzyme molecules there are in my sample. I will keep you informed!

With regard to your 340-nm increase in absorbance, even in the absence of substrate: make sure that your protein (or something else in the assay solution) is not precipitating over time, because that would give you turbidity and an increased readout at the spectrophotometer. One quick way to check for that is measuring your kinetics in the absence of NAD+.

With regarding to measure the 3 beta and 17 beta HSD enzyme activities in testicular sample from rat, As the NAD+ gets reduces to NADH by the substrate, the OD has to increase. But I am getting contrasting to that. Can anyone suggest me what could be reason to decreasing the  OD at 340 for 3 beta HSD rather than increasing in spectrophotometre? Please help...

Is your sample transparent, or does it have some turbidity? The decrease in absorbance could be an artifact caused by settling out of solids from a turbid sample that is not continually stirred.

Hello everyone,

I am working with a protein and face a similar problem during its assay. The OD at 340nm decreases continuously in the absence of the enzyme itself. My activity requires NADPH as a proton donor, GSH and Glutathione reductase and a peroxide. However in the absence of Glutathione reductase I dont see any change in the OD, but taking a control with Glutathione reductase and without enzyme gives the decrease in OD, which actually should be assumed as activity of the enzyme itself. I am not able to comprehend the reason behind this false positive result. 

Note: I usually add enzyme at the end to the already prepared mixture of above given components. My activuty assay buffer is a 50mM SPB pH7.0, 0.5mM EDTA. Purification buffer contaianing protein contains 300mM NaCl, 10% glycerol.

suggestions would be appreciated. 

thank you

Since there is a background activity without your enzyme that depends on the presence of glutathione reductase, you will have to subtract that background from the activity of the complete reaction. In other words, you need to run 2 separate reactions, one with your enzyme and one without. Then subtract the rates to get the rate due to your enzyme. You will have to make a slight change to the procedure. Do not add the glutathione reductase to the other substrates. Instead, add it at the same time as you add your enzyme, to initiate the reaction.

Thank you for the suggestion.I think thats the only way I can go with this experiment.

@ Kanti K Yadav, were you able to get satisfactory results using above method of subtracting background activity from enzyme activity?

Agree with Robson Tramontina

regards