I am trying to determine the Michaelis-Menten Constant. However, as the concentration of my peptide substrate increases, the RFU signal started to mess up and does not follow the typical kinetic which makes it hard to determine the initial rate of the reaction for the plot of Lineweaver Burk. What is the possibility that causes the fluctuation or saturation of signal? (The legend showed is the concentration of substrate while concentration of enzyme is remained constant)
Some possible explanations, not mutually exclusive, are: insufficient mixing, particulates or bubbles interrupting the light beam, and insolubility of the peptide at higher concentrations resulting in scattering of light by solids.
Adam B Shapiro is there any recommendations on solving this issue? When I tried to observe the sample after mixing, there wasn't obvious aggregates or precipitates which can be determined as insolubility but it might be because the peptide is quite hydrophobic with the fluorophore. Tried to do inner filter correction but didn't turn out to get a good correction factor or solving the issue.
The major issue at the higher concentration is the insolubility or the protein get precipitated. First, try to optimise the protein concentration, and sometimes at higher concentrations, the protein saturates, and the signal does not increase beyond a certain point. And bubble formation during addition of substrate might be one of the reason.
It looks to me like the rate of the reaction is fast during the initial 100 seconds, with not much difference between the substrate concentrations, but lower at the lowest substrate concentration than the others. After that, the rate is slower. I think what may be needed is to lower the enzyme concentration at least 10-fold so that this initial rate section of the reactions lasts longer. Then, measure the rate from about 1 -10 micromolar substrate (and zero). This should be a sufficient range of substrate concentrations to measure the kinetic constants, avoiding higher concentrations at which the substrate may not be completely soluble.
The profile shows that in higher substrate concentrations occurs flutuation of signal. It seems a solubilization problem of the substrate. Maybe , using a dilution of enzyme solution and lower substrate concentrations could useful in this case.
Thanks all for the suggestions! I tried to increase the %DMSO which really helped to solubilize the peptide but then high percentage of DMSO is not recommended for my enzyme activity. However, if I go too low enzyme concentration, the effective enzyme concentration might be much lower because it is active in dimeric form.
In my experience with substrates with Abz (0-aminobenzoyl) and Eddnp (ethylenediamine 2,4--dinitrophenyl) flanking the aminoacid sequences, the first solubilization procedure is carried out with dimethylformamide (1 mg/20 microliters; powder substrate/pure dimethylformamide). After then, the substrate solution with aqueous buffer may be made. This first solution is the stock solution, and from it you can do dilutions and no addition of dimethylformamide is necessary.