In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human α2-Macroglobulin

Complexes (2:1) of chymotrypsin with human α2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the α2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl-1-oxy results in nitroxide labeling of the active-site serine residue of the complex chymotrypsin. Electron spin resonance (ERS) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. By using 125I-labelled chyrmotrypsin, it was possible to determine the degree of covalent and noncovalent trapping of chymotrypsin under different conditions. These findings are discussed in relation to possible models of inhibition of protease activity by α2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey is the only model currently considered that can account for the present findings