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Detection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry

TitleDetection of noncovalent complex between alpha-amylase and its microbial inhibitor tendamistat by electrospray ionization mass spectrometry
Publication TypeJournal Article
Year of Publication2001
AuthorsDouglas, DJ, Collings, BA, Numao, S, Nesatyy, VJ
JournalRapid Communications in Mass Spectrometry
Volume15
Pagination89-96
Type of ArticleArticle
ISBN Number0951-4198
KeywordsANALYTICAL ULTRACENTRIFUGATION, BINDING, CRYSTAL-STRUCTURE, CYTOCHROME-C, GAS-PHASE, HEME, MYOGLOBIN, PROBING CONFORMATIONAL-CHANGES, PROTEIN PROTEINASE-INHIBITORS, RECOGNITION SITES
Abstract

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for detection of noncovalent complexes. However, detection of noncovalent protein-protein complexes is not a widespread practice and still produces some challenges for mass spectrometrists. Here we demonstrate the detection of a noncovalent protein-protein complex between alpha -amylase and its microbial inhibitor tendamistat using ESI-MS. Crude porcine pancreatic alpha -amylase was purified using a glycogen precipitation method. Noncovalent complexes between porcine pancreatic alpha -amylase and its microbial inhibitor tendamistat are probed and detected using ESI-MS. The atmosphere-vacuum ESI conditions along with solution conditions and the ratio of inhibitor over enzyme strongly affect the detection of noncovalent complexes in the gas phase. ESI mass spectra of alpha -amylase at pH 7 exhibited charge states significantly lower than that reported previously, which is indicative of a native protein conformation necessary to produce a noncovalent complex. Detection of noncovalent complexes in the gas phase suggests that further use of conventional biochemical approaches to provide a qualitative, and in some cases even quantitative, characterization of equilibria of noncovalent complexes in solution is possible. Copyright (C) 2001 John Wiley & Sons, Ltd.

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