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Labeling Histidines in Cytochrome c: An Integrated Laboratory Project
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Abstract
This four-week laboratory project uses an inorganic protein labeling reagent, chloro(2,2':6,2''-terpyridine)-platinum(II)chloride dihydrate ([Pt(trpy)Cl]Cl(2H2O), to label histidine residues of the protein, cytochrome c. The goal of the experiment is to determine the stoichiometry of labeling. Students carry out the labeling reaction and separate the unreacted labeling reagent from the labeled protein using gel-permeation chromatography. HPLC analysis of the labeled protein product mixture reveals the presence of three major peaks. These peaks are collected and analyzed by UV-vis spectrophotometry using an absorption band at 342 nm associated with the Pt(trpy) moiety and a band at 408 nm due to the heme iron of cytochrome c. UV-vis multicomponent analysis demonstrates that the three HPLC peaks correspond to unlabeled cytochrome c, monolabeled cytochrome c, and dilabeled cytochrome c.
Keywords (Audience):
Upper-Division UndergraduateKeywords (Domain):
Inorganic ChemistryKeywords (Pedagogy):
Hands-On Learning / ManipulativesKeywords (Subject):
Instrumental MethodsCiting Articles
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History
- Received: August 03, 2009
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