Photodynamic action of hematoporphyrin on yeast cells--a kinetic approach

By a technique which combines rapid mixing of cells and hematoporphyrin (HP) with a short duration of illumination, the photodynamic inactivation of yeast cells was investigated, particularly, in seeking for the information of the location of HP at the time of action. The fluence-survival curves obtained under the conditions where the reaction mixture was kept in the dark for 1 s, 60 s and even 35 min before illumination were indistinguishable from each other, indicating no interaction between cells and sensitizers took place in about 30 min in such a way that the photodynamic efficiency could be modified. It is unlikely tha HP acted intracellularly, since the protective effect of N-3 was observed at concentrations as low as 0.5 mM. Furthermore, the rate constant kp related to the protective effect of N-3 was estimated to be 1 x 10(8) M(-1)s(-1) under the assumption that (1) O2 was the active intermediate and had a lifetime of 12 microseconds under the present conditions. This value of kp is rather close to that of kq' the quenching rate constant of N-3 for (1)O2, of which the accepted value is 2 x 10(8) M(-1)s(-1) in the homogeneous aqueous system. This information, together with the absence of uptake of HP by cells and a well response of survival upon illumination to the D2O fraction of the reaction mixture, provide strong bases for the argument that direct interaction of HP with yeast cells is of minor importance in the photodynamic processes, and the photodynamic action is largely mediated by an intermediated ((1)O2) generated in bulk medium.