Abstract

Methods are described which enable accurate determination of the amounts of catecholamines covalently bound to glass beads. For isoproterenol and epinephrine these amounts are 1.68 and 1.63 µmoles, respectively, per gram of arylamine glass beads. Distillation experiments indicate that more than 80% of the tritium label released from [³H]catecholamine bound to glass is due to tritium exchange. Catecholamines labeled with tritium at position 7 should not be used to quantitate the extent of release of catecholamines coupled to glass beads, since this proton undergoes slow exchange when the [³H]catecholamine is diazotized to glass beads. Using [¹⁴C]catecholamines, the true stability of the glass bead-catecholamine complex was examined. Only 0.008% of the catecholamines are released per hour at 37°, and less than 7 x 10^-5%/hr at 4°. Under the experimental conditions for testing the biological activity of the immobilized catecholamines, less than 8 attomoles of soluble catecholamine are calculated to be released per minute into the bathing medium of the physiological preparations. Methods are also described for synthesis of amino-substituted catecholamines by reduction of the azo linkage of the catecholamine covalently coupled to solid supports. The exact structure and point of catecholamine substitution were determined by proton magnetic resonance; the results are consistent with substitution at position 6 on the catechol ring. Biological data are presented which show the amino-substituted and soluble azo-substituted catecholamines to be active when tested on isolated cat papillary muscles.