Modification of carboxyl groups of transferrin

Abstract

Carboxyl groups of transferrin were coupled with glycine methyl ester using a water-soluble carbodiimide. The iron-binding activity of the modified protein did not change until some 16 carboxyl groups had been modified. The activity then fell in proportion to the number of groups modified until no iron binding was seen with 62 carboxyl groups modified. Such a slow drop in activity was interpreted to indicate that the carboxyl groups do not directly participate in the iron-binding reaction and that the activity was lost because of a conformational change in the protein structure. Experiments involving competition of native transferrin and EDTA for iron indicated that the iron-binding activity of the former declined with increasing EDTA concentrations in a gradual manner. Similar experiments with transferrin with 15–17 carboxyl groups blocked by the glycine methyl ester showed that half of the iron-binding activity was lost with very low EDTA concentrations, remained at the 50% level over an EDTA concentration range of 0.0025–0.01 M and then commenced to decline in a gradual manner. It was concluded that the more reactive carboxyl groups are situated in the vicinity of only one of the two iron-binding sites of transferrin and that their modification then destabilizes the coordination complex. Very few if any reactive carboxyl groups are situated in the vicinity of the other iron-binding site. This finding supports the idea that the environments of the two iron-binding sites of transferrin are not identical.