The four monoiodinated forms of pork insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The procedure was rapid and extremely reproducible, resulting in total separation of the B16, B26, and A14 monoiodinated insulins from one another and from unreacted insulin. The A19 derivative eluted closely to the unreacted insulin, requiring an additional step on ion exchange chromatography for complete purification of this derivative. The receptor binding properties of the derivatives were examined in isolated rat adipocytes and hepatocytes and in cultured human IM-9 lymphocytes. The relative binding affinities of the derivatives varied in the different cell types. At tracer concentrations, the B26 derivative had the highest apparent affinity in IM-9 lymphocytes and in adipocytes, but the A14 had the greatest affinity in hepatocytes. At equivalent concentration of each tracer, IM-9 lymphocytes bound the B26 isomer 144 ± 9% (P < 0.05) and the B16 isomer 118 ± 3% (P < 0.05) as well as the 125I (A14) insulin. Isolated rat adipocytes also bound the B26 to a greater extent at tracer concentrations (117 ± 6%; P < 0.01) compared with A14, but bound B16 less than A14 (91 ± 2%; P < 0.01). Isolated rat hepatocytes bound more of the A14 derivative than either of the B-chain labels. The B26 bound 72 ± 2% (P < 0.01) and the B16 90 ± 1% (P < 0.01) as well as the A14 derivative to isolated hepatocytes.

In contrast to the results obtained at tracer concentrations of the isomers, higher concentrations of native insulin were required for 50% competitive displacement (ED50) of the B26 derivative as compared with the other two derivatives in all three cell types examined.

These results indicate a higher average affinity for the B26 isomer in the three tissues, and suggest that results obtained from tracer binding alone may not provide an accurate reflection of receptor affinity for the monoiodoinsulin isomers.

Competitive displacement of the 125I-labeled derivatives by 127I (nonradioactive) labeled homologues in the IM-9 lymphocytes also showed the B26 derivative to have the highest affinity.

The present study demonstrates that monoiodoinsulin selectively labeled on each of the tyrosines can be prepared and separated on HPLC and used to examine insulin receptor binding in different cell types. Furthermore, the data demonstrate different apparent affinities of the selectively-labeled monoiodoinsulin isomers in different cell types, suggesting variability in insulin binding properties in different tissues.

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