Isolation and characterisation of mitochondrial protein from Orthosiphon stamenius in normal and copper stress conditions

Abstract

Orthosiphon stamenius is a species of medicinal herb that can be found in tropical area. Traditionally, the leaves of this plant have been used as diuretics, treatment of rheumatism, kidney and bladder inflammation, as well as abdominal pain and gout. This herbaceous plant requires in-depth research not only for medical field, but also to understand their properties during normal and stress environment. The purpose of this study is initially to search for the most suitable technique for mitochondrial isolation from Orthosiphon stamenius leaves. Three protocols for isolation of mitochondria were performed and the mitochondrial enzyme activities were determined. The highest activity of Cytochrome c Oxidase (1.423 U/g) and the highest mitochondrial protein concentration (2.656 mg/ml) was obtained from mitochondrial isolation using a method from Millar et al. (2007). This protocol was further used for mitochondrial isolation from Orthosiphon stamenius leaves treated with different concentrations of Copper (20 ppm, 60 ppm, 140 ppm). The activity of Cytochrome c Oxidase (4.217 U/g, 4.429 U/g, 4.887 U/g) and the concentration of mitochondrial protein (2.604 mg/ml, 2.623 mg/ml, 4.887 mg/ml) showed significant increase in 20 ppm, 60 ppm and 140 ppm Copper treatment, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been conducted to check the mitochondrial protein profiles in normal and Copper treated plants. There was no obvious difference obtained in the protein profiles from normal and treated plants. There were six bands clearly visible on the gel for untreated and treated sample. Even though the protein profiles were similar, the increasing activity of Cytochrome c Oxidase and concentration of mitochondrial protein in Copper treated plants showed that plants do response under Copper stress. This study showed interconnected responses of mitochondrial activity and mitochondrial protein when exposed to different level of Copper.