Human serum albumin consists of a single polypeptide of 585 amino acid residues with 1 Trp residue. In the present work, we measured fluorescence lifetimes of the protein in both native and denatured states. The results indicate that Trp emission occurs with three lifetimes in both states. Lifetimes values and contribution to the global emission decay differ between the two states. Data are interpreted as the results of an emission occurring from three substructures of the tryptophan formed in the excited state. Two of these substructures are already present for the tryptophan free in solution. The third lifetime is the result of the interaction between the tryptophan residue and surrounding microenvironment. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the fluorophore microenvironment and on the global protein structure.
Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.
The purpose of this article is to study the relation between fluorescence decay parameters of tryptophan residue in human serum albumin (HSA) and the protein structure. HSA contains a single tryptophan residue, and thus, there will be no ambiguity on the data obtained. HSA comprises a single polypeptide of 585 amino acid residues with only one tryptophan residue. Tryptophan fluorescence is very sensitive to the local environment. Global form and structure of HSA are pH dependent. Thus, in this article, behavioral changes and fluorescence characteristics of tryptophan within HSA were studied at different pHs (2–12) and in the denatured state in the presence of guanidine hydrochloride solution. In an environment with a low pH (at pHs 2 and 3), tryptophan emits at a maximum of 330 nm. The peak position shifts to 340 nm at higher pH. Peak position values indicate no protein denaturation but a structural modification. The loss of the tertiary protein structure (complete denaturation) induces a shift in tryptophan fluorescence to 352 nm. At all pHs, tryptophan residue emits with three lifetimes. Lifetime measurements at different pHs along the emission wavelengths allowed us to differentiate the different forms of HSA. In the denatured state, tryptophan emission occurs also with three lifetimes. However, values and contributions of these lifetimes to the global emission decay differ between the native and denatured states. We have considered that fluorescence emission occurs from tryptophan substructures, each substructure is characterized by one lifetime along with its pre‐exponential. Populations of the substructures, characterized by the pre‐exponential values of the fluorescence lifetimes, are dependent on the microenvironment of the fluorophore and on the global protein structure.
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