hello,
i'm analysing enzymatic activity in lichens samples using UV-VIS spectrophotometer, for all samples i'm having an absorbance value of 4, after dilutions of 50% and 20% i'm still having the same value, i wonder what would be the signification of this result ?
Your absorbance is too high. You need to dilute your sample even more. The absorbance is Abs = log (Transm) = log (Io/I). The linear absorbance range of most spectrometers is between 0.1 and 1.
At an absorbance of 2 you are at 1% Transmittance, which means that 99% of the total light is being absorbed by the sample. That is, normally, the maximum absorbance recommended for spectroscopic measurements. Above it your signal is probably saturated and not reliable. At an Abs of 4 you are at 0.01% T. That means that your spectrum is saturated.
I would recommend that you dilute your sample much more.
Your absorbance is too high. You need to dilute your sample even more. The absorbance is Abs = log (Transm) = log (Io/I). The linear absorbance range of most spectrometers is between 0.1 and 1.
At an absorbance of 2 you are at 1% Transmittance, which means that 99% of the total light is being absorbed by the sample. That is, normally, the maximum absorbance recommended for spectroscopic measurements. Above it your signal is probably saturated and not reliable. At an Abs of 4 you are at 0.01% T. That means that your spectrum is saturated.
I would recommend that you dilute your sample much more.
Just to expand a little bit on the previous answer, "to dilute your sample much more" means that you have to dilute your sample not only to 50 % or 20 %, but at least by 3 orders of magnitude (maybe even more, since your signal was already saturated)...
As per the enzyme kinetics; when the enzyme is saturated with substrate is the maximum rate of reaction Vmax can be achieved . . ... This is usually provides information about the Km (Michaelis constant) of the enzyme, an inverse measure of affinity. For practical purpose , if your substrate concentration reaches upto saturation point. The reaction will be not follow the kinetics equation and you will get same absorption value during assay even if you change the enzyme concentration . So it is important to determine the Vmax and find the saturation concentration. You can dilute your samples untill you find the saturation points as well as Vmax of your enzyMe. I hope that will worK.
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