Oxidatively Modified Proteins in a Clinical Laboratory

Detection of Oxidatively Modified Proteins in a Clinical Laboratory 

Protein thiolation is quantified by measurement of release of protein-bound thiols under reducing conditions alternatively, modification of protein isoelectric charge due to thiolation is detected (M17, T7, T8, T10). 

Formation of carbonyl groups causes a decrease in amino group number in protein molecules. Therfore the loss in amino group content is also used as a measure of oxidative protein modification marker. The protein amino group quantitation is most commonly performed employing the fluorescamine assay. Fluorescamine reacts with primary amines to form a fluorescent product (excitation wavelength 390 nm emission at 440-500 nm) (R9). Older methods, such as employing trini-trobenzenesulfonate (H2), are less sensitive and more troublesome. 

Chlorination products (mainly chlorotyrosine) are measured by HPLC (Fig. 8) or gas chromatography-mass spectrometry. Also, 3-nitrotyrosine can be detected in protein hydrolysates by HPLC in combination with various detection systems, including UV and electrochemical detection (Cl7, C20, L23, L24, 04, S26), gas chromatography, gas chromatography-mass spectrometry (J2), electrospray mass spectrometry, and Western blotting or ELISA using antinitrotyrosine antibodies (H20, T2, V6). 

Tryptophan damage is estimated by decrease of its native fluorescence (excitation 279-298 nm, emission at 320-350 nm, depending on the microenvironment) or increase in fluorescence of its oxidation products (R9). A-Formylkynurenine emits light with maximum intensity at 454 nm when excited at 360 nm in neutral or acid medium. The fluorescence yield increases about 20-fold when the measurement is performed at pH 10.5 (excitation at 315 nm and measuring emission at 400 nm) (G32). 

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