A method for obtaining a tritium-labeled S-100 protein of high specific radioactivity (-10 Ciimmol) under mild conditions is described. The method is based on the reductive methylation of lysine residues; the labeling procedure does not appreciably alter the physical and chemical properties of S-100 protein, as measured by studies of intrinsic fluorescence enhancement, Ya binding, electrophoretic mobility, titrations of sulfydryl groups, and intramolecular crosslinking of S-100 via disulfide bond formation. Alternative labeling procedures based on chemical or enzymatic iodination with lz51 involving the use of powerful oxidiz-, ing agents, cause an irreversible oxidation of the sulfydryl groups and affect the above-mentioned properties of the S-100 protein.
Although several immunological, physical, and chemical properties of the brain-specific S-100 protein (l-4) have been described, its function remains unknown. Recent studies of the calcium-dependent effects of this protein on membrane permeability may have some important biological implications (5).
The availability of labeled S-100 would allow new experimental approaches to the study of the biological role of this protein. In particular, it would make it possible following the interaction of S-100 with organized cellular structures and with other molecules.
In our and in other laboratories, attempts have been made to label the S-100 protein by iodination with lz51 The iodination reaction occurs, . however, in the presence of oxidizing agents which affect the SH groups and, consequently, some physicochemical properties of the protein (4,6). This is to be expected, considering that according to recent studies (6) cysteine residues are certainly involved in the cation-induced conformational changes and in the DTNB-catalyzed stabilization of the quaternary structure of the protein. Moreover, iodination affects tyrosyl residues which are known from spectral and chemical studies (3) to be involved in the characteristic conformational transition induced in the S-100 protein