Intrinsic protein absorbance proteins, concentration

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

Some proteins, such as collagen and HPr, do not contain tryptophan or tyrosine residues therefore they do not intrinsically absorb light at 280 nm. In these cases, it will be necessary use either AAA, refractive index ( ) detection, backbone absorbance (<240 nm), or a colorimetric method (see below). When using refractive index detection, the incremental change in n with concentration (dn/dc, a constant analogous to a protein s extinction coefficient) must be known. The determination of dn/dc requires the generation of a standard curve ( vs. concentration) the concentration must be previously determined by different method. It quickly becomes evident how circular the determinations can be, with one method relying on the outcome of another. 

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