Some time ago Schultz and his colleagues (Schultz et al., 1972; demonstrated that myeloperoxidase, when injected daily into tumour-bearing mice in conjunction with thio-TEPA, causes a significant decrease in the rate of tumour growth. Neither myeloperoxidase nor thio-TEPA alone appeared to be effective, and retardation in tumour growth was only observed as long as treatment was continued.In vitro, myeloperoxidase in the presence of hydrogen peroxide and a halide ion exerts a potent cytotoxic activity against a variety of cell types. These include bacteria (Klebanoff & Luebke, 1965;Klebanoff et al., 1966Klebanoff et al., , 1970Klebanoff, 1967Klebanoff, , 1968McRipley & Sbarra, 1967), fungi (Lehrer, 1969;Lehrer & Jan, 1970;Diamond et al., 1972;Howard, 1973), viruses (Belding et al., 1970, and a variety of mammalian cells (Klebanoff & Smith, 1970;Edelson & Cohn, 1973;Clark et al., 1975;Clark et al., 1976;Clark & Klebanoff, 1977). Presently available evidence suggests that the toxic activity of myeloperoxidase involves the generation of oxygen radicals or similar highly reactive species (Klebanoff & Clark, 1978). Other peroxidases such as lactoperoxidase and horseradish peroxidase have similar cytotoxic properties (Oram and Reiter, 1966;Jacques et al., 1975;Reiter, 1978 In order to construct an enzyme system that would function as the peroxidase-H202-halide system in vivo we immobilized the peroxidase together with a hydrogen peroxide producing enzyme onto small solid beads. This would assure that hydrogen peroxide is continually produced in the immediate vicinity of the peroxidase. Since chloride ions are ubiquitiously distributed, no special action was considered necessary to assure the availability of halide ions. Thus, a 20g CNBractivated Sepharose-4 was suspended in 50 ml 0.1 M phosphate buffer, pH 7.0, containing 1% lysine, and the suspension was stirred overnight. The gel was then thoroughly washed with water and stirred for 10h in 5% NaCl. The gel was again washed and then filtered over a Buchner funnel. The packed gel was resuspended in 20 ml buffer and 2ml 50% glutaraldehyde was added. After 1 h stirring at room temp. the gel was washed thoroughly with buffer. The gel was then added to a solution containing 20mg horseradish peroxidase (Sigma, type VI) and 4.5 ml glucose oxidase (A. niger; Sigma, type V, 1000 U ml-1). The mixture was gently shaken overnight at 4°C and the immobilized enzymes were separated from any unbound enzymes by the procedure described by Mosbach and Mattiassen (1976). The gel was then lyophilized to dryness and the dry powder was stored at -20°until use.Our first experiments were done using Novikoff hepatoma bearing rats. Three week old SpragueDawley rats were inoculated i.p. with 0.5ml of a Novikoff hepatoma cell suspension and the tumour was allowed to develop for 5 days. Each rat was then injected i.p. with 5mg of the immobilized enzymes, suspended in 1 ml PBS containing 0.1% glucose. This treatment was repeated for 5 consecutive days. All 10 control animals died within 15 days aft...
