skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence

Abstract

The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.

Authors:
;  [1];  [2]
  1. Ames Lab., IA (United States)
  2. Massachusetts Institute of Technology, Cambridge, MA (United States)
Publication Date:
OSTI Identifier:
559860
Report Number(s):
CONF-970443-
TRN: 97:005895-0018
Resource Type:
Conference
Resource Relation:
Conference: 213. national meeting of the American Chemical Society, San Francisco, CA (United States), 13-17 Apr 1997; Other Information: PBD: 1997; Related Information: Is Part Of 213th ACS national meeting; PB: 2904 p.
Country of Publication:
United States
Language:
English
Subject:
40 CHEMISTRY; 55 BIOLOGY AND MEDICINE, BASIC STUDIES; DETECTION; ELECTROPHORESIS; FLUORESCENCE; MONITORING; POLYPEPTIDES; SPECTROPHOTOMETRY; TRYPTOPHAN; VISCOSITY; ENZYMES; PROTEIN STRUCTURE

Citation Formats

Jensen, P K, Lee, Cheng S, and King, J A. Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence. United States: N. p., 1997. Web.
Jensen, P K, Lee, Cheng S, & King, J A. Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence. United States.
Jensen, P K, Lee, Cheng S, and King, J A. 1997. "Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence". United States.
@article{osti_559860,
title = {Monitoring refolding of tailspike endorhamnosidase using capillary electrophoresis-laser induced tryptophan fluorescence},
author = {Jensen, P K and Lee, Cheng S and King, J A},
abstractNote = {The use of capillary electrophoresis equipped with laser-induced tryptophan fluorescence detection is presented for monitoring the refolding pathway of phage P22 tailspike endorhamnosidase. Upon initiation of refolding, tailspike polypeptides rapidly fold into structured monomeric intermediates with a high content of secondary structure. These monomeric species associate to form the triple-chain defined folding intermediates, the protrimers. Conversion of the protrimer into the native, sodium dodecyl sulfate (SDS) resistant tailspike protein is the rate-limiting step in the refolding pathway. Refolding kinetics and yield measured by capillary electrophoresis are in good agreement with those obtained via native gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence spectrophotometry. To enhance separation resolution between protrimer and native protein in capillary electrophoresis, the use of poly(ethylene oxide) is investigated for the introduction of a sieving separation mechanism. The increased viscosity of the electrophoresis buffer may also play a role in resolution enhancement.},
doi = {},
url = {https://www.osti.gov/biblio/559860}, journal = {},
number = ,
volume = ,
place = {United States},
year = {Wed Dec 31 00:00:00 EST 1997},
month = {Wed Dec 31 00:00:00 EST 1997}
}

Conference:
Other availability
Please see Document Availability for additional information on obtaining the full-text document. Library patrons may search WorldCat to identify libraries that hold this conference proceeding.

Save / Share: