hello,
I need to create a binding curve to measure the affinity of my enzyme to its zinc cofactor, to determine the Kd value.
I'm using prism (graphpad) and there are a lot of options under the binding option.
there are options for one or to sites- I'm not sure whether it relates to the number of catalytic sites (meaning if the enzyme is monomer or dimer) or to the number of zinc atoms bound to each enzyme unit.
I also am not sure whether it matters for the program choice whether the binding is to the metal or a ligand.
thank you,
sapir
I am not sure what you are really asking. If you have a protein P and a ligand L, then you need to separate the free and the bound ligand. This can be done for example by dialysis, ultrafiltration (DOI:10.1006/abio.1998.2854), gel filtration (DOI:10.1016/0006-3002(62)90124-5), ultracentrifugation (DOI:10.1021/bi00572a003, http://www.jbc.org/content/143/3/753.full.pdf, DOI:10.1021/bi00876a003), electrophoresis (DOI:10.1002/elps.1150050402) or by mass spectrometry (DOI:10.1006/abio.2000.4975).
The number of binding sites is determined by a Job-plot (DOI:10.1016/S0076-6879(82)87029-8).
If you have a single binding site, the binding equation is (using LaTeX notation): \( K_d = \frac{[P] [L]}{[PL]} = \frac{([P]_t - [PL]) ([L]_t - [PL])}{[PL]} \). After some arithmetic, you get the basic form of a quadratic equation, \( 0 = -[PL]^2 + [PL] (K_d + [P]_t + [L]_t) - [P]_t [L]_t \). This equation has two solution, but as there are no negative concentrations we need only the positive, which is the Langmuir-isoterm (DOI:10.1021/ja02242a004): \( [PL] = \frac{([P]_t + [L]_t + K_d) - \sqrt[2]{([P]_t + [L]_t + K_d)^2 - 4 [P]_t [L]_t}}{2} \). From this equation, you can calculate [PL] from [P]_t and [L]_t, that is, without mixing dependent and independent variables.
Of course, when there is more than one binding site, the equation becomes more complicated.
Hi dear
https://web.stanford.edu/group/solomon/Publications_files/cr9500390.pdf
as attached file
Best regards
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