Radioimmunoassay of methionine(5)-enkephalin sulphoxide: phylogenetic and anatomical distribution

A sensitive and specific radioimmunoassay (RIA) for the oxidised form of methionine(5)-enkephalin (Met(5)-Enk), Met(5)-Enk sulphoxide (Met(5)-Enk-S), has been developed. Antisera were raised in rabbits against Met(5)-Enk coupled to carrier proteins with glutaraldehyde or carbodiimide. Displacement of (125I) Met(5)-Enk bound to antiserum by Met(5)-Enk was poor, but Met(5)-Enk-S displayed good displacement suggesting that the Met(5)-Enk immunogen was oxidised to Met(5)-Enk-S and that the antisera were formed against this compound. The sensitivity of the RIA for Met(5)-Enk-S was 0.02 pmole/tube using the most sensitive antiserum. The antisera showed negligible cross-reactivity with leucine(5)-enkephalin and with both native and oxidised endorphins. Cross-reactivity was between 15% and 28% with the fragment Met(5)-Enk (2--5) sulphoxide and between 9% and 25% with D-Ala(2)-Met(5)-Enk sulphoxide. The antisera showed less than 0.01% cross-reactivity with other Met(5)-Enk fragments and naturally occurring neuropeptides. Tissue extracts were oxidised with hydrogen peroxide prior to assay. Met(5)-Enk-S immunoreactivity (IMR) was detected in brain, pituitary gland, pancreas, and intestine extracts of the rat, chicken, toad and teleost, and in cerebral-suboesophageal ganglion extracts of the snail. All tissue extracts showed parallelism in serial dilution to synthetic mammalian Met(5)-Enk-S, suggesting possible immunological identity. The results indicate that spontaneous oxidation of Met(5)-Enk immunogen occurs such that antisera are produced against the sulphoxide analogue of Met(5)-Enk, and may account for the relative insensitivity of some published RIAs using Met(5)-Enk standard. Our findings demonstrate a wide phylogenetic and anatomical distribution of Met(5)-Enk IMR.