Comparison of iodinated [Nle¹⁵]‐ and [Met¹⁵]‐gastrin₁₇ prepared by reversed‐phase HPLC

¹²⁵I‐[Nle¹⁵]‐gastrin₁₇ prepared by the iodogen method can be separated by reversed‐phase high performance liquid chromatography into two peaks, both of which elute after [Nle¹⁵]‐gastrin₁₇. Direct determination of the specific activities of the two derivatives by microbore reversed‐phase HPLC indicated that they were the mono‐ and di‐iodinated species. In contrast the two peaks obtained with [Met¹⁵]‐gastrin₁₇ iodinated under the same conditions eluted earlier, relative to the appropriate gastrin₁₇ standard, than the [Nle¹⁵]‐gastrin derivatives. Treatment of either peak with 0.75 M dithiothreitol at 56°C or 95°C resulted in progressive conversion to compounds migrating in relative positions similar to the ¹²⁵I‐[Nle¹⁵]‐gastrin₁₇ derivatives. Direct determination of the specific activity of the earlier eluting [Met¹⁵]‐gastrin₁₇ derivative before reduction indicated that it was the mono‐iodinated species. It thus appears likely that iodination of [Met¹⁵]‐gastrin₁₇ by the iodogen method results predominantly in the formation of mono‐ and di‐¹²⁵I‐[Met sulphoxide¹⁵]‐gastrin₁₇. To avoid problems arising from oxidation of the methionine residue of gastrin during iodination, the use of ¹²⁵I‐[Nle¹⁵]‐gastrin₁₇ in binding studies is therefore recommended.