Iodination of beta-melanotropin. Time course analysis of reaction mixtures by high pressure liquid chromatography and characterization of biologically active mono- and diiodo-beta-melanotropin.

D T Lambert,C Stachelek,J M Varga,A B Lerner

doi:10.1016/s0021-9258(18)34319-9

Abstract

The production of highly purified, chemically well defined, iodinated beta-melanotropin possessing full biological activity is described. beta-Melanotropin, purified by high pressure liquid chromatography, was iodinated using Iodogen as the oxidizing agent. Biological activity was recovered, following iodination, by incubating the iodinated peptide mixture in 0.75 M dithiothreitol at 37 degrees C for 18-24 h. The qualitative and quantitative effects of dithiothreitol and the changes that occurred in the iodination mixture with time were examined using high pressure liquid chromatography. This separation technique has proved to be a useful tool both for optimizing the iodination procedure and for the rapid, efficient purification of mono- and diiodinated beta-melanotropins. Amino acid analysis of these two peptides revealed modifications only of the one tyrosine residue (Tyr 5): the expected incorporation of iodine to form mono- or diiodo tyrosine. Monoiodo-beta-melanotropin had full biological activity, as measured by tyrosinase stimulation in Cloudman S91 melanoma cells, while diiodo-beta-melanotropin was an order of magnitude less active.

Highlights

The production of highly purified, chemically well and free from contaminants which might interfere with quandefined, iodinated 8-melanotropinpossessing full bio- titative binding studies or qualitativeinvestigations (e.g.aulogical activity is described. 8-Melanotropin, purified toradiography, electron microscopy)
The qualitative and quantitativeeffects substantially reduced biological activity [7,8,9,10]; a principal Atpionpohfuirmdqeerdiusiiniinfsetoiauhocdhtriaiiaoaeonttsicnahoipltdinrrqiemoouianovtiinoxfdepaltdrulmacoytrhnocsoeerdibnsdoeowtmuoh-arifeatehattuacohnsnhgdetedarisfmnaeufdopgleitrehiwottsy“owhdto.hoheienliarprsaateebstppooeeietcdptdixchda,uaeer8mrffsau-fromtiiserricniodeieneenvlogidnanepthnteattiocilghmehthdre-iozpincmcptcihnhahgreeoueestscmm.sheeeiiidowccounaaefrlilrllneeyytehd.woiiWrsdneellsyyleiindidpeaepulacfdrermettibsivea(eiado1lnlt,l0ytioho)gi.pnhgiuIechnraralieylpflasiypetuedapadcpauritrsenirotvisdocfeieiwepndderuborwre“ede5huno1ifcocx-otlthisardcba(tpheht4rlaieeoo,r8dadn,clupa1tcooeb1ifrren,icl1zgtiihe2nndea)ge, modifications only of the one tyrosine residu(e”yr 5): P-MSH, which possesses full tyrosinase-stimulating activity. the expected incorporation of iodineto form mono- or This probe should be useful in examining the interaction of diiodo tyrosine
A 3-min iodination, followed by dithiothreitol treatment, yields a mixture containifnougr main components which absorb UV light at 280 nm (Fig. la).The compcinent that elutes first is free of radioactivity and has the same retention timaes purified unlabeledP-MSH;the second component ( F )contains an amounotf radioactivity consistent with its beingmonoiodo-P-MSH, this beingconfirmed by amino acid analysis

Summary

Introduction

The production of highly purified, chemically well and free from contaminants which might interfere with quandefined, iodinated 8-melanotropinpossessing full bio- titative binding studies or qualitativeinvestigations (e.g.aulogical activity is described. 8-Melanotropin, purified toradiography, electron microscopy). The production of highly purified, chemically well and free from contaminants which might interfere with quandefined, iodinated 8-melanotropinpossessing full bio- titative binding studies or qualitativeinvestigations By high pressure liquid chromatographwya, s iodinated These conditions could be satisfied by the use of a radiolausingIodogen as the oxidizing agent.Biological activity beled hormone of high specific radioactivity, as can be prowas recovered, following iodination, by incubatingthe duced by incorporating ‘‘‘1 into the peptide. The iodinated peptide mixturein 0.75 M dithiothreitol at 37 iodination of MSH has generally resulted in a hormone with “Cfor 18-24 h. As measured by tyrosinase stimula- describethe value of high pressure liquid chromatography tion in Cloudman S91 melanoma cells, while diiodo-8- as a tool in analyzing and optimizing iodination conditions

Methods

Results

Conclusion