Spectrophotometric assay using streptavidin-alkaline phosphatase conjugate for studies on the proteolysis of polymer bead-bound peptides.
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 alpha-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 alpha-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 alpha-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.
Shin
J S
JS
Department of Chemical Technology, Seoul National University, Seoul, Korea, 151-742
Kim
B G
BG
Kim
D H
DH
Lee
Y S
YS
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D002918:Chymotrypsin / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011108:Polymers; D013053:Spectrophotometry / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D010455:Peptides; D011108:Polymers; D009580:Nitroblue Tetrazolium; D019809:Streptavidin; D000469:Alkaline Phosphatase; D002918:Chymotrypsin