CHLORAMINE-T IN RADIOLABELING TECHNIQUES .2. A NONDESTRUCTIVE METHOD FOR RADIOLABELING BIOMOLECULES BY HALOGENATION

Chloramine-T (CAT) is commonly used in the radiolabeling of bioactive molecules by halogenation. CAT may be used either as a solution or in an immobilized form (Iodobeads) to release radioactive elemental iodine or other halogens by oxidation of their salts. CAT has a very high chlorine potential, and it causes oxidative damage to sensitive substrate molecules, such as peptides and proteins. In some cases, the substrates are completely destroyed. To reduce the chlorine potential of CAT, morpholine was mixed with CAT prior to exposure to the substrates. This formed N-chloromorphholine, in situ, which readily reacted with KI to form I-2. The kinetics of the formation of N-chloromorpholine from CAT and morpholine were studied spectrophotometrically by following the disappearance of CAT at 250 nm. The reaction was found to be rapid at all pH's from 5 to 11. 1-Aminocyclohexanecarboxylic acid (a model amino acid) decomposed rapidly in the presence of CAT, but there was no decomposition in the presence of N-chloromorpholine. N-Chloromorpholine was compared to CAT solution and Iodobeads for the iodination of L-tyrosine. The formation of mono- and diiodotyrosine were followed by HPLC. On an equimolar basis (0.55 mu M), N-chloromerpholine produced a much greater yield of the mono- and diiodinated tyrosine than Iodobeads. Furthermore, decomposition products were observed when tyrosine was exposed to Iodobeads for 15 min. When a CAT solution was used at a higher concentration (5.5 mu M), a substantial amount of decomposition occurred, and the yields of the two iodinated species were very small. When a similar concentration of N-chloromorpholine was used (5.5 mu M), the yield of the iodinated species was essentially the same as that at the lower concentration (0.55 mu M), and no decomposition was observed. When the CAT alone and CAT + morpholine methods were compared for the iodination of a model peptide, leucine enkephalin, it was found that the CAT + morpholine method produced larger amounts of what appeared to be iodoleucine enkephalin and diiodoleucine enkephalin as well as less decomposition of the peptide after 2 and 15 min exposure. It is proposed that the method employing N-chloromorpholine (which is produced in situ instantaneously) to release diatomic iodine is more convenient and efficient for radiolabeling peptides and proteins than methods currently used. (C) 1995 Academic Press, Inc.