The activities of carbonic anhydrase and alkaline phosphatase in ancient human bones. Purification and characterization of outer peripheral, cytosolic, inner peripheral, and integral CA

In the present study, bone carbonic anhydrase was isolated from ancient hum an bones and its characteristic features were determined. For this purpose, the skull bone of about 3000 years age was used. The purification was perf ormed in four steps. Four different isoenzymes of CA, including outer perip heral, inner peripheral, integral, and cytosolic were purified and characte rized. Affinity chromatography using Sopharose-4B-L-tyrosyn sulfanilamide a s a support material was used in its purification. Two different methods were used for enzymatic activity determination: a) hy dratase, and b) esterase methods. Bradford and Coomassie Brillant Blue meth ods were used for protein determination. Optimal pH, temperature, and molec ular weight determinations were performed by conventional methods. The puri fication degree and the subunits, if present, were determined by SDS-PAGE. The effects of some chemicals on the enzyme were also investigated. The mos t cardinal finding was that the enzymatic activity has been found in antiqu e human bone, showing some other enzymatic activity. That the alkaline phos phatase activity has been determined in the same sample supports the findin g of carbonic anhydrase.