Analysis of the interferences in quantitation of a site-specifically PEGylated exendin-4 analog by the Bradford method

Xiaowei Qian; Hongxia Dong; Xiaojing Hu; Hong Tian; Linfeng Guo; Qingliang Shen; Xiangdong Gao; Wenbing Yao

doi:10.1016/j.ab.2014.06.009

Analysis of the interferences in quantitation of a site-specifically PEGylated exendin-4 analog by the Bradford method

Anal Biochem. 2014 Nov 15:465:50-2. doi: 10.1016/j.ab.2014.06.009. Epub 2014 Jun 19.

Authors

Xiaowei Qian¹, Hongxia Dong¹, Xiaojing Hu¹, Hong Tian¹, Linfeng Guo¹, Qingliang Shen¹, Xiangdong Gao², Wenbing Yao³

Affiliations

¹ Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China.
² Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China. Electronic address: xdgao@cpu.edn.cn.
³ Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China. Electronic address: wbyao@cpu.edu.cn.

PMID: 24953009
DOI: 10.1016/j.ab.2014.06.009

Abstract

Protein modification has been found to affect the estimation of protein concentration in some of the traditional dye-based absorbance measurements. In this work, a distinct reduction in A595 was observed during the quantitation of a PEGylated exendin-4 analogue (Ex4C) by the Bradford method and the PEGylation process was found to interfere with the measurement. Lys(12), Arg(20), and Lys(27) were further proved to be the major amino acids that functioned as dye-binding sites. The shielding effect produced by the large polymer was demonstrated to depend on the length of PEG that was used for modification.

Keywords: Bradford method; Exendin-4; Interference; PEGylation; Protein concentration; Shielding effect.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Exenatide
Humans
Peptides / analysis*
Peptides / chemistry*
Polyethylene Glycols / chemistry*
Venoms / analysis*
Venoms / chemistry*

Substances

Peptides
Venoms
Polyethylene Glycols
Exenatide