Chemical modification probes accessibility to organic phase: proteins on surfaces are more exposed than in lyophilized powders

Chemical modification of myoglobin and cutinase suspended in n-hexane by ac yl chlorides and iodine was monitored by electrospray mass spectrometry. Th e general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modific ation rates were slower for larger acyl chlorides, particularly with lyophi lized powders. About 20% of the protein molecules in lyophilized powders we re modified much more quickly than the rest, a fraction consistent with tho se exposed on the surface of the solid. It appears that access to most of t he molecules in lyophilized powders requires a very slow stage of solid-pha se diffusion. This has been neglected in previous discussion of mass transf er limitation of lyophilized enzymes in organic media, and would not be rev ealed by the experimental evidence used to dismiss it. Studies of the effec ts of particle size and dilution with inactive protein are only sensitive t o diffusion in liquid-filled pores, not through the solid phase. Slow solid -phase diffusion is not required for access to most support-adsorbed protei ns, which is probably a major contributory factor to their enhanced catalyt ic efficiency in organic media. Hydration of lyophilized proteins accelerat es chemical modification rates, as it does their catalytic activity. The ma in site of reaction of acyl chlorides in organic media is not amino groups (which are probably ion-paired), but is likely to be hydroxyl groups instea d. (C) 2000 Elsevier Science Inc. All rights reserved.