Abstract
Tris and phosphate buffer are regularly used in experimental investigations. These buffers might have radical scavenger properties toward different kinds of Reactive Oxygen Species (ROS) produced in solutions during chemical reactions like Fenton reaction and gamma radiolysis. Hydroxyl radicals (ºOH) are the most reactive and oxidizing agents having a great potential in oxidization of macromolecules like DNA and proteins. This <em>in vitro</em> study was aimed to evaluate radio-protective effects of Tris and phosphate buffer toward hydroxyl radicals generated by Fenton reaction. Hence, ºOH radicals were produced using a mixture of Hydrogen Peroxideand Ferrous Sulfate, called Fenton system. Human serum albumin (500µM) was prepared in Tris (10mM) and phosphate buffer (10mM), separately. These two samples were incubated with Fenton reaction (Ferrous Sulfate + Hydrogen peroxide) (10 mM) for 30 minutes and carbonyl groups were quantified by spectrophotometric carbonylation assay. The results of this study revealed the values of 1.04 ± 0.02 and 1.73 ± 0.03 for Tris and phosphate buffer treated samples, respectively. In conclusion, these findings confirmed that Tris buffer is a stronger radical scavenger toward ºOH radicals<strong> </strong>than phosphate buffer.<strong> </strong>
Highlights
Since several years ago, Tris and phosphate buffer have been used prevalently in most of experimental investigations due to their powerful buffering function in the range of physiological pH
Human serum albumin (HSA) is one of the most common proteins being investigated by scientists during recent decays (Kondakova, Ripa, & Sakharova, 1988; Maciazek-Jurczyk & Sulkowska, 2015; Sitar, Aydin, & Cakatay, 2013)
A spectrophotometric method for protein carbonyl assay was chosen to compare radical scavenger power of Tris versus phosphate buffer toward oOH radicals. It is clearly demonstrated in figure 1 that Tris buffer is a stronger radical scavenger than phosphate buffer toward oOH radicals at pH=7 in vitro
Summary
Tris and phosphate buffer have been used prevalently in most of experimental investigations due to their powerful buffering function in the range of physiological pH. Hydroxyl radicals are highly reactive in direction of interaction with macromolecules in physiological conditions (Cheeseman & Slater, 1993; Du & Francisco, 2008; Harman, 1992; Loizos, 2004; Pryor et al, 2006; Winterbourn, 1995) These radicals can damage proteins by creating protein backbone cleavages, making inter and intra molecular cross-links, generating carbonyl groups, conversion of free thiol groups (-SH) to other forms and so on (Anraku, Yamasaki, Maruyama, Kragh-Hansen, & Otagiri, 2001; Leeuwenburgh, Hansen, Shaish, Holloszy, & Heinecke, 1998; Plowman, Deb-Choudhury, Grosvenor, & Dyer, 2013). Human serum albumin includes 582 amino-acids with a molecular weight of 66KD jmbr.ccsenet.org
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