Journal
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
Volume 60, Issue 1-2, Pages 22-28Publisher
ELSEVIER
DOI: 10.1016/j.molcatb.2009.03.003
Keywords
Immobillized alpha-chymotrypsin; Porcine pepsin A; Magnetic particle; Phosphopeptide analysis
Funding
- Ministry of Education of the Czech Republic [MSM 0021620806, LC 06044]
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To detect phosphorylation state of pepsin, a simple and a rapid procedure for coupling of alpha-chymotrypsin to commercially available magnetic particles was elaborated. alpha-Chymotrypsin was immobilized to -CHO activated commercial magnetic particles via the protein free amino groups. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH dependence of the activity, thermo-stability, self-cleavage activity, and possibility of repeated use. The immobilized alpha-chymotrypsin was used to study the phosphorylation degree of porcine pepsin A, used as a model acidic protein and phosphoprotein. The use of enzyme immobilized to magnetic carriers has several advantages as compared with an application of soluble forms of enzymes: preferably an increased stability of enzymes, a possibility of direct use of enzyme reaction products for MALDI-TOF MS. The prepared proteolytic digest was separated using RP-HPLC and immobilized metal affinity chromatography (IMAC) with immobilized Fe(III) ions prior to MALDI-TOF analysis: the presence of phosphopeptide in porcine pepsin A was shown. (C) 2009 Elsevier B.V. All rights reserved.
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