On the labelling of oxidized cell surface membranes by acyl hydrazides

Abstract

The covalent modification of bovine erythrocyte membranes by first oxidizing cell surface sugars followed by the covalent coupling of the oxidized components with hydrazides was investigated. The aldehydic groups were introduced either chemically, by mild periodate oxidation, or enzymically with galactose oxidase. The periodate (0.1 mM) and galactose oxidase (50 units) oxidations were complete at 5 and 240 min, respectively, in phosphate-buffered saline (pH 7.4) at 30°C using 2 · 10 9 cells · ml −1. The rates of hydrazone formation between the oxidatively generated aldehyde groups and α-[ 3H]acethydrazide were also measured (pH 7.4) at 30°C. The reactions with the periodate and galactosde oxidase-treated cells went to completion in 1.5 and 6 h, respectively. Aniline had a marked catalytic effect on these rates. With 5 mM aniline under the same conditions, the periodate-treated cells reacted to completion with acethydrazide in 30 min and the galactose oxidase-treated cells in 40 min. As expected, although the rates of α-[ 3H]acethydrazide incorporation were increased with aniline, the extents of the reaction were not. Finally, the stability of the hydrazone linkages were measured. The hydrazone bonds formed when the galactose oxidase-treated cells were reacted with acethydrazide were completely stable at 30°C for 24 h whereas those formed from the periodate-treated cells were not. In the latter case roughly 50% of the membrane-bound hydrazone linkages were lost after 6 h at 30°C. The remaining 50%, however, were stable to cleavage. Thus, these sites are clearly heterogeneous in nature.