Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration.
Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay.
Guermant
C
C
Protein Chemistry Unit, Faculty of Medicine, University of Brussels, 808 Route de Lennik, Bruxelles, B-1070, Belgium.
Azarkan
M
M
Smolders
N
N
Baeyens-Volant
D
D
Nijs
M
M
Paul
C
C
Brygier
J
J
Vincentelli
J
J
Looze
Y
Y
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D003545:Cysteine / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000737:chemistry; D004151:Dipeptides / Q000032:analysis / Q000737:chemistry; D006441:Hemoglobin A / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D009113:Muramidase / Q000032:analysis / Q000737:chemistry; D010084:Oxidation-Reduction; D010940:Plant Proteins; D011192:Potassium Dichromate; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results
D000596:Amino Acids; D004151:Dipeptides; D007202:Indicators and Reagents; D010940:Plant Proteins; D011506:Proteins; D006441:Hemoglobin A; D009113:Muramidase; D003546:Cysteine Endopeptidases; C039974:caricain; D003545:Cysteine; D011192:Potassium Dichromate