INCREASED PERMEABILITY OF SUPEROXIDE-DISMUTASE AT THE BLOOD-NERVE ANDBLOOD-BRAIN BARRIERS WITH RETAINED ENZYMATIC-ACTIVITY AFTER COVALENT MODIFICATION WITH THE NATURALLY-OCCURRING POLYAMINE, PUTRESCINE

Our previous studies have demonstrated that modification of superoxide dismutase (SOD) with the naturally occurring polyamines-putrescine (P UT), spermidine, and spermine-dramatically increases the permeability- coefficient surface area (PS) product at the blood-brain barrier and b lood-nerve barrier after parenteral administration. Because of this in creased permeability, the efficient delivery of polyamine-modified SOD (pSOD) across these barriers may enhance its therapeutic usefulness i n treating ischemic neuronal degeneration, neurodegenerative disease, or even aging as an important antioxidant therapeutic strategy. Becaus e PUT-SOD had the highest PS values, SOD was modified in the present e xperiments by activating carboxylic acid groups to the reactive ester with water-soluble carbodiimide and then reacted with PUT as the nucle ophilic reagent. Preservation of SOD enzyme activity while maximizing the permeability was accomplished by adjusting the ionization of the p rotein carboxylic acid with pH. Both sodium dodecyl sulfate-polyacryla mide gel electrophoresis and isoelectric focusing analyses demonstrate d graded conversion of SOD to its polyamine-modified derivative when p erformed at different pH. Although modification at pH 4.7 resulted in only 6.6% retained SOD activity and the highest PS value (43.35 +/- 3. 81 x 10(-6) ml/g/s for the hippocampus), modification at pH 5.7 result ed in 50.1% retained activity with a PS value of 24.48 +/- 1.30 x 10(- 6) ml/g/s for nerve endoneurium and 21.95 +/- 1.62 x 10(-6) ml/g/s for hippocampus. This contrasts with a PS of 1.8-3.2 x 10(-6) ml/g/s for native SOD in nerve and various brain regions. Reaction conditions are therefore defined that titrate enzyme activity of PUT-SOD with PS cha nges in the intact animal after intravenous administration. These stud ies will allow an evaluation of the therapeutic usefulness of pSOD in animal models of neuronal degeneration.