When i study the binding interaction with metal complex and Serum protein. i have gotten the stern volmer constant as, At 20oc Ksv = 1.49x 104 and Kb = 1.49 x 104 . At 30oc Ksv = 1.18 x 104 and Kb = 2.89 x 106. At 40oc Ksv = 1.49 x 104 and Kb = 2.3 x 104.
In many papers, the i have seen that the Ksv neither increase nor decrease . but in my case from 20oc to 30oc it decrease and 30oc to 40oc it increase. even i have repeated the experiment many time i have found the same result and one more thing if i took the emission spectra of BSA (1x10-5M) with in a 5min interval i cannot get the same intensity value. for example At 40oc i got like 268,260 and 257. Should i take the average intensity for the calculation?
Is it instrumental error? or nature of the metal complex and protein interaction?
kindly help me to solve the problem and give some relevant articles.
If your fluorescence intensity is continuously falling, your sample may be equilibrating or deteriorating. All other interpretations are impossible until you determine this.
Ksv and Keq may both be temp dependent.
Why not you try to perform a florescence quenching experiment at three mentioned temperature levels 20, 30 and 40 degree Celsius? Perform the experiment with charged and uncharged quenchers. Then compare Ksv and Kq with previously obtained result obtained without quenchers. Do not forget to compensate dilution caused by adding the solution of quencher.
Just different approach to the problem
Kind regards
Hi Manoj,
Few things should be remembered before analysis of the fluorescence quenching data.
1. Incubation time with the ligand should be such to achieve equilibrium. Atleast 10 minutes incubation time should be given after addition of the ligand to the protein solution.
2. Selection of the molar ratio between ligand and protein in order to get significant quenching (~50%) in the last tube of the titration expt.
3. Regarding decreased fluorescence intensity of BSA solution, are you using the same tube to read the intensity after 5 min interval? If yes, it is wrong. You should take the same solution of BSA in 3 different tubes. Then read the fluorescence intensity. It will come out the same value.
4. Ksv and Kb should change with temperature, showing either increase or decrease with temperature.
Please check these points.
Best.
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