My metal complex neither soluble in water nor Buffer. it soluble in methanol,, CHCl3 ... but for the biological convenient i dissolved in DMSO solvent and dilution with PBS Buffer. During the fluorescence experiment (synchronous ) , in first addition i have found that the tyrosine intensity increases but decrease in next additions. i have took 1:1 ratio concentration of metal complex and BSA (1x10-6 moL concentration ). i don't know the reason for the anomalous behavior. Should we use DMSO for protein binding study . if it possible kindly help me in this doubt and give some related articles
take into account that dmso absorption increases as you go to shorter wavelengths (270-280nm).
it is not clear what additions and where.
the easiest thing to do is to add pure dmso and see for yourself.
Since the absorption peak of tyrosine (~274) is so close to the cut off for DMSO (~268), you should definitely see some effect from the DMSO just from excitation attenuation as suggested by Alexander. But there are some other effects to take into account as well. DMSO increases the quantum yield of tyrosine by a small amount (~+6%). Tyrosine is sensitive to the proximity of other amino acid residues so protein folding can have a significant effect on fluorescence in either the positive or negative depending on the residues that are near. BSA has 17 disulfide bonds that may begin to dissociate in the presence of DMSO, especially if the pH is above 7 or below 5. All of this may result in strange behavior of the fluorescence based on concentrations of DMSO. You'll need to be careful when using DMSO in protein binding studies since it is known to denature proteins (disulfide bonds) which will change the exposed residues and change the adhesion behavior. At very low concentrations (
Dear Y,
I would suggest you that an alkali or acidic aqueous solution/buffer to deal with the solubility of Tyrosine (please refer to the paper as below). It may offer you some advantages in lowering both protein degradation and background absorance in the UV region. Organic solvent is eventually not a good option for protein quantization.
http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC2140676&blobtype=pdf
Really Thanks for your answers. If it possible kindly let me know that can i use DMSO + buffer mixture for protein binding study.? of else can i use methanol + buffer mixture ?
I believe less than 10% of DMSO would be safe for the protein stability. For example, firstly dissolve the metal complex in 1 mL DMSO and then add up 9 mL of basic/acidic buffer in a 1/10(v/v) ratio.
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