Selective real-time disulfide reduction with simultaneous fluorescent labeling of a humanized anti-cocaine mAb

Abstract

A procedure for rapid screening and development of facile and limited partial reduction, with simultaneous fluorescent labeling, of a humanized anti-cocaine mAb was devised and characterized. This employed a standard soluble protein reductant, tris (2-carboxyethyl) phosphine (TCEP), paired with an alkylating agent that can be used without removal of the reductant, yielding a fluorescent adduct of the generated cysteine. This reaction can also be monitored by absorbance in a nanophotometer, allowing for rapid, simple screening of reduction and alkylation conditions using small mAb amounts and volumes. The degree of reduction is readily controllable, and yields results similar to limited reductions followed by separate labeling reactions, as recently reported using immobilized TCEP, and a less reactive phosphine reductant, triphenylphosphine-3,3′,3″-trisulfonic acid (TPPTS). The overall structure of the mAb is not perturbed, and the thermal stability of the Fab portion of the mAb, where most selective disulfide reductions occur, is only minimally decreased. The antigen (cocaine) binding and binding thermodynamics are not changed, as demonstrated by isothermal titration calorimetry. However, differential scanning fluorimetry demonstrated that the thermal stabilization of the Fab domain by cocaine binding is dramatically decreased after reductive labeling to a level of 5.44 modified cys/mAb, suggesting the importance of light-heavy chain disulfide bonds for the cocaine-induced thermal stabilization of the mAb Fab. Methods described in this work should aid in the selective reduction and labeling of disulfides in mAbs and other proteins, assisting the assignment of structural and functional importance to specific protein disulfide bonds.