I am trying to understand if, two different ways of performing enzyme binding kinetics may impact the final Kd values and if yes to what extent for slow and fast reactions.
For more context :
Densitometry couple with Gel based techniques like EMSA, immunoblotting and ELISA would rely on doing these kinetic measurements by allowing varying concentrations of the reactants and observing for equilibrium state. While kinetic measurements done using advanced methods like ITC, Stopped flow or BIA core would do real time measurements for attainment of equilibrium.
Any literature with such comparison will be highly appreciated.It would also be helpful to answer this question by keeping in mind the sensitivity of the technique used here
Please, check the following post:
https://www.researchgate.net/post/Why_would_Kd_value_vary_across_FP_ITC_and_MST_assay_significantly
Thanks Adrian for the thorough explanation.
Would you happen to know of any literature which has done a comparative analysis of this sort?
Secondly, would you also comment on the same keeping in mind the sensitivity of an assay, for example if I am to perform densitometric studies using radiolabelled substrates in one case and another using fluorescence stains (like ETBr) the detection limits would be completely different. How can this discrepancy be addressed ?
When the concentrations employed in different techniques are quite different, because of technique limitations or restrictions, then the practical range for reliable affinity determination is different. For example, it is assumed that in ITC the practical range for Kd determination is from 100 microM to 1 nM, because protein concentration in the calorimetric cell is usually in the order of 5-50 microM, but in fluorescence titrations you may determine Kd values well below 1 nM.
Error margins for Km are much larger than those for Vmax. Thus, you should first do a t-test for the differences of the means, given their standard deviation. Only significant differences are worth discussing further.
I must sadly say that enzyme kinetic study should be performed by the uniquely quantitative HPLC-photometric method (please see file; J Chrom B Rat BIN LIP Km).
Further, since protein and glycoprotein change the affinity to substrate or ligand via the total chemical structure of them, old chemical idea of Kd should not be utilized in biochemistry. Km should be utilized instead.
Furthermore, nature and also biochemical world has no Normal distribution or Gaussian distribution (Gauß-Verteilung), and Non-parametric statistical analysis should be utilized (please see file; Dr. Terentyeva Urine BIN). Therefore, I have proven that Fucoidan/Fucoidin is surely the effective anticancer drug by using Non-parametric statistics (Mann-Whitney’s U test) and Bernoulli trial/Binomial trial of Dr. Jacques Bernoulli (please see file; Rat DEN Np-Fuco).
Furthermore, precise, sensitive and reliable protein determination should be performed by the unique HPLC-Surf-SEC protein determination method (please see file; HPLC-Surf-SEC Protein determination method).
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