Calibration of Oxygen-Dependent Quenching of the Phosphorescence of Pd-meso-tetra (4-Carboxyphenyl) Porphine: A Phosphor with General Application for Measuring Oxygen Concentration in Biological Systems

Abstract

Oxygen-dependent quenching of phosphorescence is a function of the frequency of collision between the phosphor and molecular oxygen and of the efficiency of energy transfer during these collisions. Thus, quenching is dependent on the rate of diffusion of the phosphor and its molecular environment. For measurements in biological samples, the Pd-porphyrin is bound to serum albumin, and this provides a uniform microenvironment for the phosphor which is relatively unaffected by changes in the pH and ionic composition of the medium. Calibration of the phosphor is of particular value because it is absolute, i.e., the calibration is valid independent of the laboratory and the time of measurement. This paper reports the calibration constants determined for Pd-meso-tetra (4-carboxyphenyl) porphine, as measured by two independent methods: by stoichiometric titration of the oxygen with ascorbate in the presence of ascorbate oxidase and by comparison with a high-accuracy oxygen electrode. The measurements were carried out in a specially designed thermostatted vessel in which the oxygen electrode and phosphorescence lifetime measurements of oxygen were made simultaneously. The calibration constants for the oxygen-dependent quenching of the phosphorescence of Pd-meso-tetra (4-carboxyphenyl) porphine were determined as a function of albumin concentration, ionic strength in medium, pH, and temperature.