The Effect Of Ph On Enzyme Activity Assignment Sample

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Protein Purification And The Effect Of Ph On Enzyme Activity Using Alcohol Dehydrogenase

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Introduction

Protein purification is essential for understanding the characteristics and properties of a particular protein. Usually, proteins are not found in nature in a single and purified state. Therefore, for conducting any protein-related biochemical research, a particular protein can be extracted from either a complex mixture or plant tissue or animal tissue. In protein-related research works, the extraction and purification of protein can be conducted with several protein purification techniques such as gel electrophoresis techniques, fractionation techniques and chromatography etc. Here, in this paper, the protein is purified by using Ion-exchange Chromatography. Ion-exchange chromatography is a chromatographic separation process. Generally, this chromatographic method is used to understand deamination as well as succinimide formation (Bernal et al. 2018). This process is based on the net or surface charge of a particular protein. There are two types of ion-exchange chromatography. Anion exchange (the protein is negatively charged at the certain pH value, generally higher, of a particular buffer) and cation exchange (the protein is positively charged at the certain pH value, generally lower, of a particular buffer). In ion-exchange chromatography, protein molecules are separated on the basis of their charge. By using a strong ionic solution the molecules are washed and separated. The ionic solution is passing through the column and the molecules are separated according to their charges. Enzymes are proteinaceous in nature and made up of polypeptide chains (composed of amino acids). Here, this work is performed with alcohol dehydrogenase. It is a zinc enzyme and the oxidation of primary and secondary alcohol is catalysed by this. In ion-exchange chromatography, the separation is affected by the changes in pH value. The surface charge of the protein and the function of pH value are related to each other as the charge of the amphoteric molecules is determined by the pH value of the buffer. Raising or reducing the pH value of the buffer can alter the charge of the molecules. The Isoelectric point or PI of a protein is a certain pH value of a protein where the protein becoming neutral means carrying no net charge. If this happens in ion-exchange chromatography, the reaction cannot be forwarded. The effect of pH on the enzyme (alcohol dehydrogenase) activity is determined with a spectrophotometric quantitative assay that is mainly of two types- the endpoint assay and kinetic assay. Every enzyme has an optimum pH and altering the pH value can affect the enzyme activity. By measuring the changes in light intensity that are either absorbed or scattered by the reactive solution, the relation between pH and enzyme activity can be determined.

Results

 

Absorbance (280 nm)

Wash 1

0.000

Sample flow-through

1.108

Wash 2

0.112

Eluate

1.222

  1. The principle of ion-exchange chromatography states that the protein molecules move through the column according to their net electric charge that depends on the used pH value. With the cation exchanger, the protein molecules that contain more negative net charge run faster as well as elute earlier and this can happen simultaneously with the anion exchanger. This report is done with the absorbance at 280 nm and from the above report, it can be said that after the first wash, none of the protein molecules is absorbed and eluted. After sample flow through the column and second wash, the eluted rate increases (the higher the protein concentration in the sample, the greater amount of light is absorbed) that denote the maximum protein concentration (alcohol dehydrogenase) in the sample is present.
  2. In cuvette 1, the buffer solution with pH 7.0 is taken. In cuvette 2, the buffer solution with a pH value of 8.5 is taken. The concentration value is measured with the absorbance of 340 nm. 0.2 ml of alcohol dehydrogenase is added into the solution of both cuvettes at the interval of 15 sec. It can be stated from the results that the enzyme activity increases with time and the concentration of alcohol dehydrogenase (graph 1 and 2). But the catalytic power of alcohol dehydrogenase is greater in pH 8.5 (graph 3) though the enzyme is activated lately.

Discussion

It is essential to purify protein while conducting any biochemical experiment that is related to protein as, in nature, any purified form of the protein cannot be found. Protein can be easily found in plant tissue or animal tissue or any complex solution. To understand the effectiveness and nature of a particular protein, the protein purification process is necessary. There are various techniques for purifying the protein. The main advantages of protein purification techniques as a particular protein can be isolated from the technique, providing maximum recovery yield, high accuracy, greater matrix tolerance, and rapid separation, high precision and high selectivity (Javedet al. 2018). Alcohol dehydrogenase comes from the dehydrogenase enzyme family that helps to interconversion ketones or aldehydes and alcohols by the reduction of nicotinamide adenine dinucleotide to NADH (Liu et al. 2018). Naturally every enzyme is proteinaceous in nature thus by measuring the concentration of a particular protein in a solution, the researcher can know how much protein is present in the solution. Protein purification is conducted by ion-exchange chromatography. The alcohol dehydrogenase is extracted from the chromatography column with Tris buffer and thus the protein concentration is measured with the UV spectrophotometer. Protein concentration is always measured at 280 nm as the protein molecules can absorb the UV light with absorbance majorly at 280 nm. The peptide bonds of proteins are generally responsible for this. This procedure helps to know the amount of protein or protein concentration of the given sample. Every enzyme has its optimum pH that denotes that the activity of a particular enzyme becomes greater in that pH (Juturu and Wu, 2018). Here, the effect of pH on the activity of alcohol dehydrogenase is determined by the spectrophotometric assay. Two values of pH (pH 7.0 and 8.5) were taken whereas the optimum pH of alcohol dehydrogenase is 10. From the result, it can be stated that at the exact optimum pH or near the optimum value, the enzyme activity increases whereas lower or greater the optimum value decreases the enzyme action.

References

Journals

Bernal, C., Rodriguez, K. and Martinez, R., 2018.Integrating enzyme immobilization and protein engineering: an alternative path for the development of novel and improved industrial biocatalysts. Biotechnology advances36(5), pp.1470-1480.

Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M.H., Riaz, M., Afzal, M., Kouser, A. and Nadeem, H., 2018. Bacterial lipases: a review on purification and characterization. Progress in biophysics and molecular biology132, pp.23-34.

Juturu, V. and Wu, J.C., 2018. Heterologous protein expression in Pichia pastoris: latest research progress and applications. ChemBioChem19(1), pp.7-21.

Liu, Q., Zheng, J., Sun, W., Huo, Y., Zhang, L., Hao, P., Wang, H. and Zhuang, M., 2018.A proximity-tagging system to identify membrane protein–protein interactions. Nature methods15(9), pp.715-722.

Tao, J., Zhao, Y.Q., Chi, C.F. and Wang, B., 2018. Bioactive peptides from cartilage protein hydrolysate of spotless smoothhound and their antioxidant activity in vitro. Marine drugs16(4), p.100.

Winkler, M., 2018.Carboxylic acid reductase enzymes (CARs). Current opinion in chemical biology43, pp.23-29.

Zamora-Sillero, J., Gharsallaoui, A. and Prentice, C., 2018. Peptides from fish by-product protein hydrolysates and its functional properties: An overview. Marine Biotechnology20(2), pp.118-130.

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