Ascertaining Free Histidine from Mixtures with Histidine-Containing Proteins Using Time-Resolved Photoluminescence Spectroscopy

The use of photoluminescent probes for differentiating free amino acids from biomolecules containing the same amino acids is challenging. Photoluminescent probes generally present similar emission spectra when in the presence of either free-amino acids or protein containing those same amino acids. Probes based on cyclometalated iridium(III) complexes Ir(L)(2)(sol)(2) (where L is 2-phenylpyridine, 2-(2,4-difluorophenyl)pyridine, or benzo[h]quinolone, and sol is a solvent molecule) present long-lived emission when bound to histidine. This emission is tuned by the microenvironment around the complex and therefore its lifetime is different for free histidine (487 ns) than from histidine-containing proteins such as bovine serum albumin (average lifetime > 700 ns). As a proof-of-concept we demonstrate that free histidine can be discerned from a mixture with histidine-containing proteins by using time-resolved photoluminescence decays. In the presence of multiple sources of histidine, iridium(III) probes display a multiexponential decay, which can be fitted by nonlinear least-squares methods to separate the different components. Because the pre-exponential factor of the 487 ns lifetime is proportional to the concentration of free histidine, we can use it to assess the amount of free histidine in solution even in the presence of proteins such as bovine serum albumin. We also show that iridium(III) probes displaying different photoluminescence maxima can be produced by modifying the ancillary ligands of the metal complex.