TY - JOUR
T1 - Poly(ethylene glycol)-supported enzyme inactivators. Efficient identification of the site of covalent attachment to α-chymotrypsin by PEG-TPCK
AU - Schering, Christine A.
AU - Zhong, Boyu
AU - Woo, Jonathan C.G.
AU - Silverman, Richard B.
PY - 2004
Y1 - 2004
N2 - A new methodology utilizing an enzyme inactivator covalently attached to poly(ethylene glycol) (PEG) is described in which the PEG affords facile and mild quantification, isolation, and identification of the site of enzyme inactivation. As proof of concept, the known affinity labeling agent for α-chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), was linked to PEG. The synthesis of the PEG-bound inactivator PEG-TPCK was carried out in good yields using standard solution-phase chemistry. Inactivation of α-chymotrypsin with PEG-TPCK was monitored via UV-vis spectroscopy in aqueous conditions, which resulted in less than 3% remaining activity, indicating that 97% of the α-chymotrypsin was covalently modified with PEG-TPCK. The MALDI-TOF mass spectrum showed only one new peak that was distinct in shape and corresponded to the mass of PEG-TPCK-α-chymotrypsin. Following proteolytic digestion, the PEG-TPCK-peptide was easily discernible from the rest of the digest in a HPLC trace because of its characteristic prolonged retention time and broad polymer shape. MALDI-TOF MS was used to determine the mass of the PEGylated peptide. Without prior removal of the PEG, the amino acid site to which PEG-TPCK covalently bound was determined via Edman sequencing. In comparison to other methods, the PEG-supported inactivator system is significantly cheaper and safer than the synthesis of radiolabeled compounds; furthermore, isolation of the PEGylated peptide is milder and more selective than standard affinity binding columns. Edman sequencing provides an exact determination of the site of inactivator covalent attachment without extensive, tedious LC-MS analysis of a complex peptide mixture. The method described here could be applied to a variety of enzymes as an alternative to current techniques.
AB - A new methodology utilizing an enzyme inactivator covalently attached to poly(ethylene glycol) (PEG) is described in which the PEG affords facile and mild quantification, isolation, and identification of the site of enzyme inactivation. As proof of concept, the known affinity labeling agent for α-chymotrypsin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), was linked to PEG. The synthesis of the PEG-bound inactivator PEG-TPCK was carried out in good yields using standard solution-phase chemistry. Inactivation of α-chymotrypsin with PEG-TPCK was monitored via UV-vis spectroscopy in aqueous conditions, which resulted in less than 3% remaining activity, indicating that 97% of the α-chymotrypsin was covalently modified with PEG-TPCK. The MALDI-TOF mass spectrum showed only one new peak that was distinct in shape and corresponded to the mass of PEG-TPCK-α-chymotrypsin. Following proteolytic digestion, the PEG-TPCK-peptide was easily discernible from the rest of the digest in a HPLC trace because of its characteristic prolonged retention time and broad polymer shape. MALDI-TOF MS was used to determine the mass of the PEGylated peptide. Without prior removal of the PEG, the amino acid site to which PEG-TPCK covalently bound was determined via Edman sequencing. In comparison to other methods, the PEG-supported inactivator system is significantly cheaper and safer than the synthesis of radiolabeled compounds; furthermore, isolation of the PEGylated peptide is milder and more selective than standard affinity binding columns. Edman sequencing provides an exact determination of the site of inactivator covalent attachment without extensive, tedious LC-MS analysis of a complex peptide mixture. The method described here could be applied to a variety of enzymes as an alternative to current techniques.
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U2 - 10.1021/bc049940p
DO - 10.1021/bc049940p
M3 - Article
C2 - 15264852
AN - SCOPUS:3242745982
SN - 1043-1802
VL - 15
SP - 673
EP - 676
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 4
ER -