Oxidation of Free L-histidine by tert-Butylhydroperoxide

Purpose L-histidine, a commonly used buffer for protein formulations, has the potential to oxidize and form multiple byproducts. Previous studies were performed using metal catalyzed oxidation with Fe²⁺ or Cu²⁺. We re-examined the oxidation of L-histidine under conditions more appropriate to protein...

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Bibliographic Details
Published in:Pharmaceutical research 2010-03, Vol.27 (3), p.447-456
Main Authors: Mason, Bruce D, McCracken, Melissa, Bures, Edward J, Kerwin, Bruce A
Format: Article
Language:English
Subjects:
Amino acids
Biochemistry
Biological and medical sciences
Biomedical and Life Sciences
Biomedical Engineering and Bioengineering
Biomedicine
Catalytic oxidation
Cations - chemistry
Cellular biology
Chemical compounds
General pharmacology
Histidine - chemistry
Medical Law
Medical sciences
Metals - chemistry
Oligopeptides - chemistry
Oxidation-Reduction
Pharmaceutical sciences
Pharmaceutical technology. Pharmaceutical industry
Pharmacology. Drug treatments
Pharmacology/Toxicology
Pharmacy
Proteins
Research Paper
tert-Butylhydroperoxide - chemistry
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Summary:Purpose L-histidine, a commonly used buffer for protein formulations, has the potential to oxidize and form multiple byproducts. Previous studies were performed using metal catalyzed oxidation with Fe²⁺ or Cu²⁺. We re-examined the oxidation of L-histidine under conditions more appropriate to protein formulations. Methods Solutions of free L-histidine, protected from light, were initially reacted with tert-butylhydroperoxide and the products analyzed by UV absorption spectroscopy, reversed phase HPLC and mass spectrometric analysis and NMR. Experimental parameters investigated were oxidizing agent, pH, temperature, metal ion and metal chelator content. Results The initial reaction produced a number of known products, along with an unknown product that was identified as 4(5)-imidazolecarboxaldehyde. The reaction was highly pH and oxidizing-agent specific. The product was not observed at pH 5.0 or below, while there was a dramatic increase for reactions carried out at pH 6.0 or above. Addition of FeSO₄ to the reaction dramatically increased the amount of 4(5)-imidazolecarboxaldehyde produced, while addition of the metal chelators EDTA or DTPA completely inhibited the reaction. Conclusions The presence of oxidants and trace concentrations of metal ions in high purity L-histidine solutions results in the formation of 4(5)-imidazolecarboxaldehyde which has the potential to covalently modify proteins.
ISSN:0724-8741
1573-904X
DOI:10.1007/s11095-009-0032-y