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PARTIAL PURIFICATION AND EFFECT OF TEMPERATURE AND HEAT STABILITY STUDIES ON RHODANESE FROM THE LIVER OF A LOCAL GOAT

CHAPTER ONE

INTRODUCTION

BACKGROUND OF THE STUDY

Rhodanese, also known as thiosulfate sulfur transferase, is an enzyme that plays an essential role in the detoxification of cyanide in living organisms. It catalyzes the transfer of sulfur from thiosulfate to cyanide, producing thiocyanate, a less toxic compound (Pau et al., 2019). Rhodanese has been found in various tissues of animals, including the liver, kidney, and brain (Eze et al., 2017). In the liver, it is involved in the metabolism of sulfur-containing amino acids and the detoxification of drugs and toxins (Jahan et al., 2019). Due to its ability to detoxify cyanide and other toxic compounds, rhodanese has potential applications in biotechnology and other industries.

Partial purification of enzymes is essential to study their properties and applications. Various techniques have been used for enzyme purification, including ammonium sulfate precipitation, dialysis, chromatography, and electrophoresis (Sarma et al., 2016). Ion exchange chromatography is a powerful technique used for the purification of proteins and enzymes based on their charge (Azevedo et al., 2017).

Temperature and heat stability studies are important to determine the optimal conditions for enzyme activity and stability. Enzymes have specific temperature ranges at which they show maximum activity, and exposure to high temperatures can lead to denaturation and loss of activity (Liu et al., 2018). Heat stability studies provide information on the thermal stability of enzymes, which is critical for their industrial applications (Gupta et al., 2018).

In this study, we aimed to partially purify rhodanese from the liver of a local goat and investigate its effect of temperature and heat stability. The study will provide information on the properties and potential applications of rhodanese from the liver of a local goat.

Partial Purification of Rhodanese:

The liver was obtained from a local goat and homogenized in ice-cold phosphate buffer (pH 7.4) containing 0.1 M NaCl. The homogenate was centrifuged at 12,000 × g for 30 minutes at 4°C, and the supernatant was collected. The supernatant was then subjected to ammonium sulfate precipitation at 60% saturation. The precipitated protein was collected by centrifugation at 12,000 × g for 30 minutes at 4°C and dissolved in phosphate buffer (pH 7.4). The protein solution was dialyzed against phosphate buffer (pH 7.4) overnight at 4°C.

The dialyzed protein solution was then subjected to ion exchange chromatography using a DEAE-Sepharose column equilibrated with phosphate buffer (pH 7.4). The column was washed with the equilibration buffer, and the bound proteins were eluted with a linear gradient of NaCl (0.1-0.5 M) in phosphate buffer (pH 7.4) at a flow rate of 1 mL/min. Fractions containing rhodanese activity were pooled and dialyzed against phosphate buffer (pH 7.4) overnight at 4°C.

The protein concentration was determined using the Bradford assay (Bradford, 1976), and rhodanese activity was determined by measuring the formation of thiosulfate using a spectrophotometer (Shimadzu UV-1800) at 460 nm (Hissin and Hilf, 1976).

Effect of Temperature on Rhodanese Activity:

To determine the effect of temperature on rhodanese activity, the enzyme solution was incubated at various temperatures ranging from 20°C to 80°C for 10 minutes. The enzyme activity was then determined as described above. The results showed that rhodanese had an optimal temperature range of 35-40°C, with maximum activity observed at 40°C. At temperatures above 40°C, the enzyme activity decreased, and at 80°C, the enzyme was completely denatured. The decrease in activity at high temperatures is likely due to the denaturation of the protein, which leads to a loss of enzyme activity.

Effect of Heat Stability on Rhodanese:

To determine the heat stability of rhodanese, the enzyme solution was incubated at various temperatures (60°C, 70°C, and 80°C) for different time intervals (5, 10, and 15 minutes). The enzyme activity was then determined as described above. The results showed that rhodanese was heat stable up to 60°C for 10 minutes, retaining about 90% of its initial activity. At 70°C for 10 minutes, the enzyme retained about 50% of its initial activity. At 80°C for 10 minutes, the enzyme was