Hi Neeta

I think you need to optimize the pH (which I suspect) or temperature or both that could cause late reaction, have you desalted your protein against the assay buffer if so then try to incubate it in 37 C or higher, what was the pH you have used? it might not the right one for this enzyme, please bear in mind that the buffer itself might not the good one however I would investigate this after pH & Temp.

Hope this helps 

Because of the long incubation time being used, make sure you have a no-enzyme blank so that you can subtract the absorbance caused by spontaneous hydrolysis of the substrate.

Lyophilization can be damaging to some proteins, causing them to lose activity. I would recommend that you store your purified protein in solution in small aliquots at -80oC or in liquid nitrogen, with 20% glycerol as a stabilizer.

Hi Abdulhadi

i used HEPES buffer of pH 6.4, 6.8 and 7.2 to carry out reaction.Thank you for answer.I will increase the temperature for reaction.

Thank you Adam for answer.

Try lower pH such of 4, 4.5, 5 & 5.5 not sure if HEPES can be used for such pHs? you may need to use another buffer double check this.

Good luck

How does the rate of hydrolysis with your purified enzyme compare with the rate you initially saw with crude extracts?  Are you losing activity somewhere along the purification process, or did you start out with low activity to begin with? The pH and temperature comments above are good ideas, but it is also possible that you don't have much activity to begin with, in which case no degree of optimization will help.  If that is the case, you may need a more concentrated preparation. This can be attained by ultrafiltration concentration. After the lyophilization step, it may also be useful if you simply redissolve in a smaller volume.

1. Make sure the reaction is enzyme catalyzed.

Use a control with not protein a incubated for the same time under identical conditions. Compare the difference in color formation.

2. Ma sure the pH of the medium does allow you to "see" de 4-nitophenolate ion (the 4-nitrofenol is colorless). Usually, reaction should either be performed at alkaline pH (10 or higher), but if the pH range of activity and stability of your enzyme is below this value, you may need to stop the reaction using  0.1 N NaOH and, if needed SDS, to ensure complete halt of enzyme catalysis.

3. Make sure the substrate concentration is enough. Increase substrate 10 times, if you see an improvement, your assay does not have a saturating substrate concentration, you need to explore the reaction as a functions of substrate concentration, determine Km and employ a concentration of substrate at least 10Km, preferably 100Km (that depends also on solubility and cost), for your routinary assay.

4. increase protein concentration in the assay. The enzyme catalysis should be proportional to enzyme concentration, unless some uncommon phenomena are taking place in your cuvette (enzyme instability, substrate instability, and undetected inhibitor present, or so on, look the Segel's Enzyme Kinetics classic text book for further possibilities explaining no linear response).

5. Make sure the incubation temperature is below the limit were your enzyme becomes unstable. Unless your enzyme is thermostable, the so called  "optimum temperature" is a feature of the assay, not of your enzyme. The theoretical curve of reaction rate should keep growing, until the enzyme starts loosing activity by thermal denaturation. That is to say, at the so-called "optimum assay temperature", your enzyme is  loosing activity, at a detectable rate, then if you increase assay time the activity loss increases exponentially and the so-called optimum moves downwards by several degrees. It is always more convenient to keep the temperature at values were your enzyme stays active for several hours, or preferably days. If your assay is going to last 24h this is a highly relevant factor.

6. Make sure there are not contaminants in your sample compromising the enzyme (i.e. proteases, heavy metals, reactive chemicals susceptible to nucleophilic attack by thiols or amines, or others).

best wishes

Rogelio

Add sodium carbonate solution, 100mM, high pH (10-12) will make the yellow become more evident (but it will probably stop your enzimatic reaction, just add it when you finished).

It will help you to read the sample, but, of course, if you could just add more protein would be better.

Increase the temperature is not always the best choice if you don't know if it can denaturate your enzyme.

Also, search if those buffers aren't inhibiting your reaction (it happens), and, if you can, see if there is another substrate that your enzyme has more activity (higher Kcat and Lower km)

Good luck