A deoxygenation system for measuring protein phosphorescence

An inexpensive and quick deoxygenation system for measuring protein phosphorescence is described. Oxygen was first reduced to less than 1 ppb from nitrogen or other inert gas by passing through an oxygen trap. The oxygen-free gas was routed through stainless steel tubing directly into the sample compartment of the phosphorimeter. Flexible tubing, coupled to the stainless steel tubing, was run through the septum of a cuvette sealed with a gray butyl rubber lyophilization stopper. The flexible tubing allowed for manipulation of the cuvette during alternate cycles of vacuuming and nitrogen equilibration. Utility of the system was demonstrated by measuring the phosphorescence lifetimes of N-acetyl-L-tryptophanamide, alkaline phosphatase, human serum albumin, and recombinant human serum albumin. Phosphorescence lifetimes of 2 ms for N-acetyl-L-tryptophanamide, almost double that previously reported, were routinely achieved while a lifetime of 1.84 s was obtained for alkaline phosphatase, well within the reported range of 1.5-2s. Human serum albumin, which contains a single tryptophan, showed a biexponential decay with lifetimes of 4.33 and 17 ms, in contrast to previous reports of a biexponential decay with rates of 0.2 and 0.9 ms. Recombinant human serum albumin was even more striking with lifetimes of 4.60 and 68.2 ms. The data are explained based on the recently published X-ray crystallographic structure of human serum albumin. The simplicity and reproducibility of the system should make this technique practical for most biochemical labs.