Project Materials

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 thiosulphate sulphur transferase, is an enzyme involved in the detoxification of cyanide in living organisms. It facilitates the transfer of sulphur from thiosulphate to cyanide, resulting in thiocyanate, a less hazardous molecule (Pau et al., 2019). Rhodanese has been discovered in numerous animal tissues, including the liver, kidney, and brain (Eze et al., 2017).

It is responsible for the metabolism of sulfur-containing amino acids as well as the detoxification of medicines and poisons in the liver (Jahan et al., 2019). Rhodanese’s capacity to detoxify cyanide and other harmful substances makes it potentially useful in biotechnology and other industries.

Enzyme partial purification is required for research into their characteristics and applications. Enzyme purification procedures include ammonium sulphate precipitation, dialysis, chromatography, and electrophoresis (Sarma et al., 2016). Ion exchange chromatography is an effective technology for purifying proteins and enzymes according to their charge (Azevedo et al., 2017).

Temperature and heat stability experiments are necessary to find the best conditions for enzyme activity and stability. Enzymes have certain temperature ranges where they are most active, and high temperatures can cause denaturation and loss of activity (Liu et al. 2018).

Heat stability studies provide information about enzyme heat stability, which is important for industrial applications (Gupta et al., 2018).

The purpose of this study was to partially purify rhodanese from a local goat’s liver and investigate its effect on temperature and heat stability. The study will provide information on the properties and potential applications of rhodanese derived from the liver of a local goat.

Partial purification of Rhodanese:

The liver was extracted from a local goat and homogenised in ice-cold phosphate buffer (pH 7.4) with 0.1 M NaCl. The homogenate was centrifuged at 12,000 × g for 30 minutes at 4°C. The supernatant was then collected. The supernatant was then precipitated with ammonium sulphate 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 dialysed with phosphate buffer (pH 7.4) at 4°C overnight.

The dialysed protein solution was then passed through ion exchange chromatography on a DEAE-Sepharose column equilibrated with phosphate buffer (pH 7.4).

The column was washed with equilibration solution, and the bound proteins were eluted using 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 with rhodanese activity were combined and dialysed against phosphate buffer (pH 7.4) overnight at 4°C.

The protein concentration was evaluated using the Bradford assay (Bradford, 1976), and rhodanese activity was determined by detecting thiosulphate production at 460 nm with a spectrophotometer (Shimadzu UV-1800) (Hissin and Hilf, 1976).

The Impact of Temperature on Rhodanese Activity:

To see how temperature affected rhodanese activity, the enzyme solution was incubated at temperatures ranging from 20°C to 80°C for 10 minutes. The enzyme activity was then assessed using the methods outlined above.

The results showed that rhodanese had an ideal temperature range of 35-40°C, with maximum activity reported at 40°C. At temperatures higher than 40°C, enzyme activity declined, and at 80°C, the enzyme was entirely denatured. The decrease in activity at high temperatures is most likely due to protein denaturation, which results in the loss of enzyme activity.

Effects of Heat Stability on Rhodanese:

To test rhodanese’s heat stability, the enzyme solution was incubated at three different temperatures (60°C, 70°C, and 80°C) for five, ten, and fifteen minutes.

The enzyme activity was then assessed using the methods outlined above. The results revealed that rhodanese was heat stable for 10 minutes at 60°C, keeping around 90% of its original activity.

After 10 minutes at 70°C, the enzyme retained roughly half of its initial activity. At 80 degrees Celsius for 10 minutes, the enzyme was