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Development of glutaraldehyde‐crosslinked chymotrypsin and an in situ immobilized enzyme microreactor with peptide mapping by capillary electrophoresis
Author(s) -
Ghafourifar Golfam,
Fleitz Antoine,
Waldron Karen C.
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200663
Subject(s) - glutaraldehyde , chemistry , chymotrypsin , chromatography , capillary electrophoresis , immobilized enzyme , reagent , substrate (aquarium) , myoglobin , microreactor , proteolytic enzymes , casein , peptide , trypsin , enzyme , biochemistry , organic chemistry , catalysis , oceanography , geology
Immobilized proteolytic enzymes present several advantages over their soluble form, not the least of which is suppression of autoproteolysis peaks even at high enzyme‐to‐substrate ratios. We have made immobilized chymotrypsin by directly crosslinking it with glutaraldehyde to produce polymeric particles. Digestion of two model substrates using the particles was followed by CE peptide mapping with detection by UV absorbance or LIF . Results showed that autoproteolysis was highly suppressed and that different storage conditions of the particles in the short term (24 h) did not affect digestion of denatured BSA . As well, the chymotrypsin particles were indifferent to the presence of fluorescein groups on a casein substrate. Glutaraldehyde crosslinking of chymotrypsin inside a fused silica capillary column to make an immobilized enzyme reactor ( IMER ) was achieved in a series of reagent addition and washing steps, entirely automated using a commercial CE instrument. Digestion of myoglobin in the IMER for 30 min at 37°C followed by peptide mapping by CE ‐ MS of the collected digest allowed identification of 17 chymotryptic peptides of myoglobin, or 83% primary sequence coverage.