The reaction with 2-hydroxy-5-nitrobenzyl bromide (HNB) is a common covalent modification of tryptophan. It results in several products which have been described by classical physico-chemical methods. To improve the understanding of the HNB-modified tryptophan structure, we synthesized a model peptide containing one tryptophan only, modified it by HNB, and analyzed the product by MALDI-TOF mass spectrometry. Surprisingly, several multi-modified products (up to 5 HNB moieties per one tryptophan) were identified. the influence of HNB concentration and pH on the degree of modification was also analyzed. In addition, a splitting of modified tryptophan peaks in MALDI-TOF spectrum was described; most probably, this effect is a common MALDI artifact of nitro-aromatic compounds which facilitates the identification of HNB-modified tryptophan by MALDI-TOF MS significantly.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.bbrc.2003.11.004 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Thermodynamic analysis of protein folding and stability using a tryptophan modification protocol.
Anal Chem
July 2014
Department of Chemistry, Duke University, Durham, North Carolina 27708, United States.
Described here is the development of a mass spectrometry-based covalent labeling protocol that utilizes the reaction of dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNSB) with tryptophan (Trp) residues to measure protein folding free energies (ΔG(f) values). In the protocol, the chemical denaturant dependence of the rate at which globally protected Trp residues in a protein react with HNSB is evaluated using either a matrix assisted laser desorption ionization time-of-flight analysis of the intact protein or a quantitative, bottom-up proteomics analysis using isobaric mass tags. In the proof-of-principle studies performed here, the protocol yielded accurate ΔG(f) values for the two-state folding proteins, lysozyme and cytochrome c.
View Article and Find Full Text PDFPhotocaged variants of the MunI and PvuII restriction enzymes.
Biochemistry
April 2011
Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes.
View Article and Find Full Text PDFRole of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.
Biol Pharm Bull
September 2006
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased.
View Article and Find Full Text PDFPurification and characterization of a novel L-2-amino-Delta2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli.
Appl Microbiol Biotechnol
September 2006
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan.
L-2-Amino-Delta2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits.
View Article and Find Full Text PDFPurification and characterization of exo-beta-D-glucosaminidase from Aspergillus fumigatus S-26.
Protein Expr Purif
January 2006
Glucosamine Saccharide Materials National Research Laboratory, Department of Agricultural Chemistry, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea.
An extracellular 104 kDa exo-beta-d-glucosaminidase was purified and characterized from the culture supernatant of Aspergillus fumigatus S-26, which showed exceptionally strong chitosanolytic enzyme activity. The purified enzyme showed optimum pH of 3.0-6.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!