I don't know why, or whether, the sulfate group increases brightness. Sulfate groups are certainly not required for fluorescent dyes to have high quantum yield. One thing that the sulfate group is good for is increasing water solubility of the dyes. This is a very valuable feature, because many fluorophores are quite hydrophobic and cause insolubility of the proteins they are attached to.

Hey Adam,

Thank you for your answer!

Alexa Fluor 488 only differs to TMR by the two sulfate groups, so i assumed, that the sulfate modifications (which are present in all Alexa variants) have a impact on the brightness in general.

"The Alexa Fluor® 555 dye—with spectral properties and quantum yield nearly identical to those of tetramethylrhodamine (TRITC) —produces brighter, more photostable conjugates. These conjugates are ideal for imaging and other applications requiring increased sensitivity and environmentally insensitive fluorescence detection."

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/fluorophores/tritc-dye.html

The brightness of the Alexa dye therefore isn't due to greater fluorescence quantum yield. It is due to less quenching of the fluorescence in the conjugate. This is probably because the sulfate groups repel each other and any negative charges on the surface of the protein and increase the hydrophilicity of the dye, reducing static quenching from dye molecules sticking to each other and to the protein surface. The Alexa dye also seems to suffer less from photobleaching, suggesting that the sulfate groups affect the photochemical processes that lead to fluorophore damage.

Dear Tim,

prior to answer your interesting question :

please correct your question as follows:

Alexa dyes have SULFONATE GROUP AND NOT SULFATE!!

If you search in the web (Google scholar) , you must have the right nomenclature with 2 sulfonate groups for alexa 488 p.e.

If you don 't correct your question, people can think that you are not familiar with chemistry.

Thank you in advance

JRG