Spectrophotometric assay using streptavidin-alkaline phosphatase conjugate for studies on the proteolysis of polymer bead-bound peptides

jong shik shin, B. G. Kim, D. H. Kim, Y. S. Lee

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Abstract

A simple method for indirectly measuring the concentration of biologically available peptides on the surface of polymer beads synthesized for the peptide or nonpeptide library is developed. To identify very small differences in the amount of cleaved peptides after α-chymotrypsin treatment, the so-called 'shaving' process, a system of biotin and streptavidin-alkaline phosphatase conjugate was used. To this conjugate, 5- bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (NBT) are coupled consecutively, to carry out alkaline phosphatase reaction and color reaction, respectively. The proteolytic action of α-chymotrypsin on surface peptides located on polymer beads was detected and cleaved peptides were quantified spectrophotometrically by analyzing unreacted NBT. Using this method, the enzymatic 'shaving' process of surface peptides on polymer beads can be optimized by examining the effects of the morphology of polymer beads and the length of spacers on how easily enzymes such as α-chymotrypsin can access the surface of polymer beads. Quantitative understanding of molecular interactions on the bead surface will give us very useful information in designing polymer bead and bead-bound oligopeptide structures for the peptide or nonpeptide library.

Original languageEnglish
Pages (from-to)9-13
Number of pages5
JournalAnalytical Biochemistry
Volume236
Issue number1
DOIs
Publication statusPublished - 1996 Apr 5

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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