Quantitative Analysis of Molecular Oxygen Using Palladium Porphyrins
Quantitative Analysis of Molecular Oxygen Using Palladium Porphyrins
A simple optical method for the quantitative determination of dissolved oxygen under physiological conditions is described. The technique involves measurement of room temperature phosphorescence from palladium(II) porphyrins. Such emission is rare for large organic molecules, and its long lifetime, together with the favourable absorption characteristics of the porphyrin receptor, ensure that the phosphorescence can be resolved easily, even in heterogeneous media. Phosphorescence yields and lifetimes are shown to be highly sensitive to the concentration of dissolved oxygen, but do not depend upon the nature of the environment, and can be used separately or together to determine oxygen levels within a biological substrate. This technique should be applicable to all areas of clinical, medicinal and biomedical chemistry.
In order to facilitate development of novel anti-cancer agents it is necessary to be able to monitor their in-situ reactivity with molecular O2, since activated forms of O2 (for example superoxide ions, hydroxyl and peroxyl radicals, singlet molecular O2) are believed to be responsible for the initiation of the chemotherapeutic process. This requires analytical determination of dissolved O2 concentrations under physiological conditions. Several experimental methods have been devised to obtain such information, including membrane polarographic detectors (1), oxygen-dependent fluorescence quenching (2), ESR active spin-labels (3), chemiluminescence (4), and phosphorescence quenching (5). Each of these approaches has its merits and disadvantages but, in terms of universal application and simplicity of operation, the luminescence quenching method is the most attractive. Because a long-lived triplet excited state will always be very much more sensitive towards O2-quenching than the corresponding short-lived singlet excited state, the phosphorescence quenching technique is to be preferred over the fluorescence approach. However, very few dyes are known that are biocompatible, stable and easily derivatised and which exhibit room temperature phosphorescence. Recent work has shown that palladium(II) porphyrins exhibit moderately intense phosphorescence in solution at room temperature, and this is quenched by O2(5). We have since found that the phosphorescence lifetime of the porphyrin can be used to give a direct measurement of the concentration of dissolved O2 in the solution. Palladium(II) porphyrins, therefore, could be used as molecular probes for determination of dissolved O2 concentrations in a wide range of environments, including biomaterials.
Discussion and Results
Photophysical Studies in Water
As an illustration of the proposed technique, we present recently obtained results describing the luminescence properties of a water-soluble palladium(II) porphyrin (PdP) in dilute aqueous solution. The porphyrin used for this study was palladium(II) tetrakis (4-sulphon-atophenyl)porphyrin (PdTSPP), the structure of which is given overleaf. This compound shows prominent absorption bands in the visible region, as shown, see Figure 1. Excitation of PdTSPP in N2 -saturated aqueous solution gives rise to the emission spectrum given in Figure 2. Both fluorescence and phosphorescence emission can be observed under such conditions. Time-resolved luminescence studies allow resolution of the two emissions since fluorescence (τ f = 0.1 ns) is very much shorter lived than phosphorescence (τ p = 1 ms). The two spectra are well separated, with minimal overlap, and the relative ratio of their maximum intensities can be measured easily. Under these conditions, the yield of total luminescence is reasonably high and can be measured with any commercial spectrofluorimeter.
Fluorescence is unaffected by the presence of molecular oxygen, whereas both phosphorescence intensity and lifetime are quenched by added O2. As shown in Figure 3, there is a linear correlation between the rate constant for decay of triplet PdTSPP, measured by laser flash photolysis methods, and the concentration of dissolved O2. For the same solutions, there is a corresponding correlation between the ratio of phosphorescence-to-fluorescence maximum intensities and the concentration of dissolved O2, Figure 4. These latter two plots can be used to determine the concentration of dissolved O2 in an unknown solution by comparing values measured for the unknown solution with the calibration plots.
Identical effects are observed with other water-soluble PdP derivatives and with water-insoluble PdP compounds dissolved in organic solvents. In all cases, the phosphorescence lifetime and total emission spectrum can be used to give a quantitative determination of the concentration of dissolved O2 in the system.
The studies outlined above have shown that dilute solutions of palladium(II) porphyrins (PdP) exhibit both fluorescence and phosphorescence at room temperature. The fluorescence yield remains independent of the concentration of dissolved O2, since the fluorescence lifetime is too short for diffusional quenching, but the phosphorescence yield (φ p) decreases progressively as the concentration of dissolved O2 increases. Under the same conditions, there is a corresponding decrease in the phosphorescence lifetime (tp); in the absence of O2 the lifetime is about 1 ms. These quenching effects follow Stern-Volmer kinetics
where φ p′ and φ p (or τ p′ and τ p) refer to phosphorescence yields (or lifetimes) recorded in the absence and presence of O2, respectively, and kQ is the bimolecular rate constant for quenching the triplet excited state by O2. Standard values representing the absence of O2 are obtained by saturating the solution with N2 and all other solutions were saturated with known mixtures of O2/N2(6). The concentration of dissolved O2 in an unknown solution is determined simply by comparing the observed τ p and φ p values with the calibration curve.
In aqueous solution τ p was found to be independent of the concentration of PdP, solution pH, ambient temperature, ionic strength and light intensity over wide ranges. The phosphorescence lifetime, therefore, gives an accurate measure of the concentration of dissolved O2. By contrast, φ p is not an absolute value but depends markedly upon concentration of PdP and light intensity. These same parameters affect fluorescence and phosphorescence to an equal degree, however, so that the ratio of the yields of phosphorescence and fluorescence can be used to determine accurate concentrations of dissolved O2, since the fluorescence yield can be used as an internal standard.
The above studies were extended to include determination of the concentration of dissolved O2 in organic solvents, micelles, liposomes, vesicles, viscous media and inside the pockets of serum albumins. These studies used a range of PdP derivatives of differing hydrophobic/hydrophillic character. The luminescence properties of each PdP were determined, as above, and their reaction with molecular O2 was quantified using laser flash photolysis methods. The effects of PdP concentration, temperature, medium, added reagents, O2 concentration and dye stability were monitored in order to establish the ability of the technique to determine meaningful O2 concentrations under such conditions.
The PdP derivatives were used subsequently to stain a variety of biomaterials, including membranes, mitochondria, macromolecular proteins, DNA, and both healthy and infected intact cells. The in-situ measurements were rendered difficult by the high levels of light scattering and the poor light transmitting properties inherent with such samples. It is desirable, therefore, to synthesise porphyrin derivatives that absorb and emit at long wavelengths where biological tissue is relatively transparent and light scattering is minimised. Thus, the luminescence properties of palladium(II) phthalocyanines, naphthalo-cyanines and “expanded porphyrins”, which should absorb and emit in the near infrared region, will be evaluated in further studies. Also, it is important to ensure that the probe molecules do not perturb the biomaterial or induce photodestruction of the medium. These studies will be described in full in a later paper.
M. Hitchman in “Measurement of Dissolved Oxygen”, Wiley-Interscience, New York, 1978
N. Opitz D. W. Lubbers Adv. Expt. Med. Biol., 1984, 180, 261
W. K. Subczynski J. S. Hyde Biophys. J., 1984, 45, 743
R. Oshino, N. Oshino, M. Tamura, L. Kobinlin-sky B. Chance Biochim. Biophys. Acta, 1972, 273, 5
J. M. Vanderkooi, G. Maniara, T. J. Green D. F. Wilson J. Biol. Chem., 1987, 262, 5476
A. Mills, A. Harriman G. Porter Anal. Chem., 1981, 53, 1254
We thank the Texas Advanced Technology Program for financial support of this work. The Center for Fast Kinetics Research is supported jointly by the Biotechnology Resources Division of the NIH and by the University of Texas at Austin. Palladium(II) tetrakis(4-sulphonatophenyl) porphyrin can be purchased from Midcentury Chemicals, Posen, Illinois.