Cao G, Cutler R G
Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institute of Health, Baltimore, Maryland 21224, USA.
Arch Biochem Biophys. 1995 Jun 20;320(1):106-14. doi: 10.1006/abbi.1995.1347.
A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This hypothesis is based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent loss of enzyme function. This hypothesis is also supported by the report of a novel effect of N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. Here we specifically study the method that was used to measure reactive protein carbonyls in tissues. This method uses 2,4-dinitrophenylhydrazine (DNPH) and includes a washing procedure. Our results indicate that reactive protein carbonyls in normal crude tissue extracts cannot be reliably measured by this method, although it does reliably measure reactive carbonyls in purified proteins which have been oxidatively modified in vitro. The nucleic acids in tissues could be a major problem encountered in the assay. Using the streptomycin sulfate treatment combined with a dialysis step, we were successful in removing most nucleic acids from a crude tissue extract, but then the reactive carbonyl level in the crude tissue extract was too low to be reliably measured. This streptomycin sulfate treatment procedure, however, had no effect on the reactive carbonyl measurement of an oxidized protein sample. The unwashed free DNPH was another major problem in the assay because of its very strong absorption around 370 nm, where reactive carbonyls were quantitated. Nevertheless, on using the procedure described in the literature to measure total "reactive carbonyls" in rat liver and gerbil brain cortex, no change with age or PBN treatment was found. Then, we investigated a HPLC procedure which uses sodium dodecyl sulfate in the mobile phase but this was also found to be unsuitable for the reactive protein carbonyl assay in tissues.
目前一种解释衰老过程的假说认为，动物组织中氧化蛋白的积累与衰老有关。这一假说基于一系列报告，这些报告显示蛋白质羰基含量随年龄增长而增加，酶功能也随年龄增长而丧失。N-叔丁基-α-苯基硝酮（PBN）具有逆转这些随年龄变化的作用，这一报告也支持了该假说。在此，我们专门研究了用于测量组织中反应性蛋白质羰基的方法。该方法使用2,4-二硝基苯肼（DNPH），并包括一个洗涤步骤。我们的结果表明，这种方法无法可靠地测量正常粗组织提取物中的反应性蛋白质羰基，尽管它确实能可靠地测量体外氧化修饰的纯化蛋白质中的反应性羰基。组织中的核酸可能是该检测中遇到的一个主要问题。通过硫酸链霉素处理结合透析步骤，我们成功地从粗组织提取物中去除了大部分核酸，但随后粗组织提取物中的反应性羰基水平过低，无法可靠测量。然而，这种硫酸链霉素处理程序对氧化蛋白质样品的反应性羰基测量没有影响。未洗涤的游离DNPH是该检测中的另一个主要问题，因为它在37 nm左右有很强的吸收，而反应性羰基正是在此处进行定量的。尽管如此，使用文献中描述的方法测量大鼠肝脏和沙鼠大脑皮层中的总“反应性羰基”，未发现随年龄或PBN处理有变化。然后，我们研究了一种在流动相中使用十二烷基硫酸钠的高效液相色谱法，但发现该方法也不适用于组织中的反应性蛋白质羰基检测。
