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

D.Gareth Rees, Peter J. Halling

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20% of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main 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 instead.
LanguageEnglish
Pages549-559
Number of pages10
JournalEnzyme and Microbial Technology
Volume27
Issue number8
DOIs
Publication statusPublished - 1 Nov 2000

Fingerprint

Chemical modification
Powders
Membrane Proteins
Proteins
Chlorides
Diffusion in liquids
Molecules
Myoglobin
Polypropylenes
Particle Size
Silicon Dioxide
Hydration
Hydroxyl Radical
Dilution
Mass spectrometry
Catalyst activity
Mass Spectrometry
Mass transfer
Particle size
Ions

Keywords

  • chemical modification
  • enzyme accessibility
  • organic media
  • lyophilized powders
  • immobilized enzymes
  • low-water biocatalysis

Cite this

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title = "Chemical modification probes accessibility to organic phase: proteins on surfaces are more exposed than in lyophilized powders",
abstract = "Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20{\%} of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main 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 instead.",
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AU - Halling, Peter J.

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N2 - Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20% of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main 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 instead.

AB - Chemical modification of myoglobin and cutinase suspended in n-hexane by acyl chlorides and iodine was monitored by electrospray mass spectrometry. The general rate of modification was always much faster for protein adsorbed to supports (silica or polypropylene) than for lyophilized powders. Modification rates were slower for larger acyl chlorides, particularly with lyophilized powders. About 20% of the protein molecules in lyophilized powders were modified much more quickly than the rest, a fraction consistent with those exposed on the surface of the solid. It appears that access to most of the molecules in lyophilized powders requires a very slow stage of solid-phase diffusion. This has been neglected in previous discussion of mass transfer limitation of lyophilized enzymes in organic media, and would not be revealed by the experimental evidence used to dismiss it. Studies of the effects of particle size and dilution with inactive protein are only sensitive to diffusion in liquid-filled pores, not through the solid phase. Slow solid-phase diffusion is not required for access to most support-adsorbed proteins, which is probably a major contributory factor to their enhanced catalytic efficiency in organic media. Hydration of lyophilized proteins accelerates chemical modification rates, as it does their catalytic activity. The main 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 instead.

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