Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time T-M for HSA labeled with OX063 is 6.3 mu s at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.