did you run a blanks without substrate and/or enzyme? does the substrate hydrolyse gradually in your buffer? Is your enzyme active? at present it sounds like you need to go to the lab and do some control experiments. if you already did, provide some more detail on your experiment otherwise we can only suggest reasons more broadly.

Silas Mellor

Thank you for the response.

This graph shows the absorbance of different concentrations of the enzyme (0-175 nM) with 40 uM of the substrate (Nitrocefin)

as you can see, the blank`s absorbance (0 nm enzyme + 40 uM substrate) is constant by the time, and The enzyme is active.

the enzyme concentration that I used in Michaelis - Menten is 20nM with different concentrations of Nitrocefin.

Do you think I need to use higher concentration of the enzyme (e.g 40 nM) or less ?

Also, What a time setting (read per time) that should be set is preferable to calculate Vo ? ( e.g one read every 10 sec, or 20 sec, or every one minute)

Ok, looking at your progress curves a few things spring to mind. Firstly, V0 is initial rate and should be taken from the first linear part of the curve, before it starts to level off. This is in practice somewhat arbitrary, but if you do this for your experiment here you should get different inital rates.

However, in your case this brings us to another issue, which is that, particularly in the higher substrate tests, your reaction is essentially over before you start measuring, so you will not get accurate initial rate estimates with this data. I would reduce the enzyme concentration or lower the temperature to get curves that look linear for longer (you need to slow down the reaction). Ideally, you highest substrate concebtration curve should look something like your lower concentrations do now.

edit: i missed the fact that your curves show varying enzyme not substrate. Showing your varying substrate progress curves might be more informative here.

Silas Mellor

Thanks for your excellent understanding.

This graph shows the absorbance curves of 20 nM enzyme + different concentrations of the substrate as mentioned in the question.

Ok, it does look like there is a bit of change but it's slight. I would suggest you try to go lower in substrate concentration to see if you are already well above Km with your lowest concentrations. If that is the case, your rates will not show a very dramatic difference with substrate as you're already approaching the flattest part of the Michaelis Menten curve. Usually if i have no idea about the kinetic parameters of my enzyme i will try out a wide range of concentrations and do a fit with that to figure out where i need to add data points. Usually you want your substrate concentrations to cover the whole curve, with your points being roughly equally distributed above and below the Km. Ideally you should go up to 10-100 times Km but thats not always practically feasible.

It looks like your enzyme is stable for a long time (is that really minutes?), so your measurement intervals are probably OK, but you could try measuring more frequently so you will have more data points in the initial portion of the progress curve.

Silas Mellor

Oh sorry, the time scale in the previous graph is (Read per second) not minute.

In this graph, I used low concentrations of nitrocefin. (time scale is Read per minute)

Well i think you've got the answer there. Your higher concentrations are probably well above Km already, so i would focus on concentrations

Silas Mellor

Thank you very much for your answers.

Next experiments, I will use (0.05, 1, 3, 5, 10, 20, 30, 40)uM of the substrate + 10 nM of the enzyme. And repeat the same substrate concentrations scale with 20 nM of the enzyme.

Your data are still curved. Reduce the enzyme concentration to slow the reaction. Alternatively, decrease the time between measurements (if feasible) to get more points in the linear region. Given the noise in the data, the use of non-parametric fitting to determine v, and of the "direct plot" to determine Km and Vmax, is advisable. Consult a statistician if you are unsure what I am talking about.