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Analysis Of Cells For Biologically Important Macromolecules

Scientific report structure of spectrophotometry investigation into cell lines

Date : 27/11/2020

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Ali

Uploaded by : Ali
Uploaded on : 27/11/2020
Subject : Biology

Analysis of cells for biologically important macromolecules

Abstract

Biologically important macromolecules, such as proteins and carbohydrates are vital to the function of a multicellular organism. Protein is used to repair tissue and make enzymes and hormones. Carbohydrates are the main source of energy in eukaryotic multicellular organisms. potato tuber and bovine liver is rich in Carbohydrates and proteins respectively. By lysing potato and liver and forming a homogenate solution, the macromolecules contained in the cells are suspended for analysis. Analysis includes both qualitative and quantitative investigation, to detect both the presence and quantity of macromolecules in the cell samples. Qualitative test includes the iodine test for starch, quantitative test for protein includes the Biuret test, using a spectrophotometer, protein and carbohydrate standards with added Biuret reagent to draw up a calibration curve and determine the protein concentration of the liver and carbohydrate concentration of the potato. No colour change was detected for the starch test for bovine liver, and a blue black colour change was detected for the potato tuber, confirming the presence of starch. The absorbance value found for bovine liver was 0.92 at a wavelength of 595 nm and 0.04 at a wavelength of 540 nm, confirming 9.65 g L-1 of protein and 0.02 g L-1 of carbohydrates are present. The absorbance value found for potato tuber was 0.01 at a wavelength of 595 nm and 0.96 at a wavelength of 540 nm, confirming 0 g L-1 of protein and 10 g L-1 of carbohydrates are present.

Introduction

Carbohydrate storage provides multicellular organisms with energy for cellular respiration. Carbohydrates are mainly stored as Glycogen a source of amylopectin and amylose. Their properties as branched and liner structures respectively allow for effective breakdown into monosaccharides and disaccharides, glucose and fructose (Fredriksson, 1998). Amylopectin contains branched chains of glucose that are broken down by carbohydrase when needed, Amylose is a straight chain monosaccharide that can also be broken down by carbohydras when needed. Proteins such as enzymes and other biomolecules are vital to the function of a eukaryotic, multicellular organism, the structure of the liver allows for various functions, the main purpose is the processing and removal of toxic substances. The quaternary structure of proteins and the various folds in the protein molecules allow for the complex structure that a liver needs in order to process toxins. The importance of this experiment is applied to nutritional treatment knowing the concentrations of macromolecules in various tissue types in plants and animals gives doctors and dieticians a better understanding of the food needed for a balanced diet. This is important for a doctor to give dietary consultation to a patient in order to ensure better health. If a patient suffers from Kwashiorkor, a form of malnutrition due to the lack of protein, a doctor assigns a diet that is high in protein amongst other treatment, to ensure the patient is treated back to a condition of good health (NHS, 2016). Other applications include the extraction of important biomolecules once this experiment identifies a tissue type with a high concentration of a particular macromolecule, a more cost efficient method of extracting that biomolecule in high yield is achieved (Ishida, 1983). The aim of this experiment is to determine the differences in the two contrasting tissue types bovine liver and potato tuber and their varied concentrations of macromolecules protein and carbohydrates.

Method

Cell homogenate was prepared by lysing two separate cell samples potato tuber and bovine liver. 0.5 g of bovine liver was cut into millimetre sized pieces, 10 ml of 0.1% SDS/1 mM EDTA isolation buffer was added to lyse and homogenate the sample. The sample was ground into a fine homogenate. The homogenate was transferred to a 15 ml centrifuge tube, and left in a water bath at 90 C for 2 minutes in order for the proteins to go into solution. The homogenate was then centrifuged at 1,000 RPM for 1 minute in order to remove tissue fragments from solution. The liquid phase supernatant was then transferred to a test tube for analysis. This was repeated for 0.5 g for potato tuber carbohydrates were the macromolecule being dissolved into solution. Qualitative analysis for Starch was performed by adding 2 drops of 0.1 M of iodine solution and observing a colour change. Blue-black colour change indicates the presence of starch. The Biuret test was performed as a quantitative test for proteins. 3 ml of protein standards at concentrations of 0, 1, 2.5, 5 and 10 g L-1 were added to 5 separate test tubes. 3 ml of bovine liver homogenate was added to a 6th test tube and 3 ml of potato tuber was added to a 7th test tube. 3 ml of Biuret reagent was added to all test tubes and left for 10 minutes to allow for the colour to develop A 595 nm wavelength was used in a spectrophotometer, the samples were evenly distributed into cuvettes. A calibration curve of standard concentration against absorbance was drawn up from the standards in order to determine the concentration of protein in both of the homogenate samples. 1 ml of carbohydrate standards at concentrations of 0, 1, 2.5, 5 and 10 g L-1 were added to 5 separate test tubes. 1 ml of liver homogenate was added to a 6th test tube and 3 ml of potato homogenate was added to a 7th test tube. 5 ml of distilled water and 3 ml of dinitrosalicylic acid reagent was added to each test tube and covered with parafilm to prevent evaporation. The test tubes were left in a water bath at 90 C for 8 minutes, poured into cuvettes and measured in a spectrophotometer at a 540 nm wavelength A calibration curve of standard concentration against absorbance was drawn up from the standards in order to determine the concentration of carbohydrates in both of the homogenate samples.

Results

The starch test showed a blue-black colour change for potato tuber homogenate. The starch test did not show a colour change for bovine liver homogenate. THE STANDARD CONCENTRATIONS WERE REPRESENTED BY A CORRESPONDING ABSORBANCE OBTAINED FROM A SPECTROPHOTOMETER. THE ABSORBANCE OF THE HOMOGENATES ARE REPRESENTED THROUGH A VALUE GIVEN BY THE SPECTROPHOTOMETER. THE CONCENTRATIONS WERE FOUND THROUGH USING THE FORMULA OF THE GRAPH. THE CALIBRATION CURVE OF BSA AND NDSA ARE REPRESENTED BY A LINEAR TREND-LINE. THE EQUATION OF THE GRAPH IS CALCULATED AS AN AVERAGE OF THE TREND-LINE, AND THE R2 VALUE IS SHOWING ACCURACY WITHIN THE 99TH PERCENTILE.

Discussion

The bovine liver contained the highest concentration of protein between the two homogenates (refer to table 3). The absorbance for the bovine liver homogenate at a wavelength of 595 nm was 0.92, when compared to the calibration curve of BSA standards using the line equation, a concentration of 9.65 g L-1 was given. A study by Johansson and Borg found 12.5 ng of catalase in bovine liver for the processing of hydrogen peroxide. (Johansson and Borg, 1988) This is significantly higher than any other tissue in mammals as the main of the functions of the liver is the processing and removal of toxic substances. The main function of the liver is to process toxins that enter the body and remove harmful substances. This is achieved through enzymatic processes. Cytochrome P450, CYP2B6 is responsible for the metabolism of a variety of drugs and toxins in the body (Lang et al., 2001). Both potato tuber and bovine liver contained carbohydrates and only the bovine liver contained protein. The potato tuber contained the highest concentration of carbohydrates (refer to table 3). Carbohydrates are stored in liver cells as glycogen, humans store carbohydrates more effectively in the liver than other animals such as rats (Bj rntorp et al., 1978). As the study suggests the liver is an effective storage unit for carbohydrates, as less carbohydrates are stored in adipose tissue, however, this depends on the species of mammal and it s adipose tissue. The bovine liver contained a small concentration of carbohydrates as bovines contain more adipose tissue, where the carbohydrates are stored. Bovine liver is efficient at catalysing leucine and valine (Gade et al., 1981). This study by Gade shows that the bovine liver is 60% less efficient at catalysing N-formylmethionine, hence there will need to be a larger concentration of -N-Acylamino acid hydrolase as well as other proteins and amino acids in order to ensure the safe removal of toxic substances. The potato tuber s function is to store carbohydrates in the form of amylose and amylopectin (McCready et al., 1943). Starch in potato tubers are broken down through heat and shearing (Svegmark et al., 1991), this is achieved through enzymatic processes, by use of sucrose synthase. This therefore means there are proteins available in potato tubers (Junker, 2004), however the results of this experiment do not support this claim, as protein was not found in the potato tuber. Therefore, proteins found in potato tuber are only enzymes, and are available in low concentration that was undetectable in the concentration range of this experiment. Both bovine liver and potato tuber are adapted for specialised carbohydrate storage. The results express accurate scientific data, as cited by various sources. The regression line for BSA and NDSA is 0.9987 and 0.9921 respectively, showing accuracy to the 99th percentile. The starch test successfully identified the presence of starch in the potato tuber homogenate, however, the starch test was unable to show a colour change for the bovine liver homogenate. This is due to a low concentration of glycogen, a mixture of amylopectin and amylose, which can be identified by the spectrophotometer. When Iodine solution was added to the homogenate containing non-reducing sugars, a reaction occurred whereby the amylopectin was stained purple and amylose was stained a deep blue colour, this gave the dark colour that confirmed the presence of starch (Smith et al., 1979). However when the concentration of starch is very low, there is a slight colour change that is undetectable by the human eye. The applications of this experiment would be useful in biochemistry, as knowing the exact concentration of macromolecules in tissue samples will determine their function. Further tests can be done to determine which enzymes, biomolecules and proteins are present in the tissue sample, giving a more accurate idea of the function of the tissue sample. This is particularly important in medicine, as finding a tissue sample that is abundant in a particular protein, biomolecule or hormone makes for more cost efficient extraction. Dieticians use information about the macromolecule and micromolecule content in food to give an accurate diet plan for patients, to ensure good health.

Conclusion

The experiment successfully identified the different concentrations of macromolecules in the two different tissue types with an acceptable level of accuracy. The results of this experiment are supported by various previous studies that show similar trends and results. Problems in the method included lysing the tissue into a homogenate, as not breaking the cells down enough would leave the macromolecules behind in the solid phase. This gives the supernatant a lower concentration of macromolecules than expected, which can give inaccurate results. This is a human error that was avoided by using a mortar and pestle to lyse the cells into a fine paste. The equipment error in pipettes was reduced by using accurate and calibrated micropipettes. Another problem in the method was the potential loss of NDSA when the homogenate was left in the water bath. To avoid this problem from affecting the results, a temperature of 90 C was used, and the time was restricted to 8 minutes, giving enough time for the colour to develop. Parafilm was also used to prevent loss of NDSA through evaporation. Liver tissue contains a high concentration of protein, potato tissue contains a high concentration of carbohydrates. These tissue samples are adapted to the function of the eukaryotic cell they are part off. Liver tissue processes and removes toxins in the body, hence it contains a high concentration of enzymes, cytochromes and muscle tissue. Proteins allow for a complex three dimensional structure, so the folds and intermolecular bonds in the protein allow the liver tissue to have a complexity that allows it to process toxins. Carbohydrates stored in the form of glycogen in potato tissue, this is a simple structure made of granules of starch, the starch is more dense near the centre of the potato, this allows for easier digestion by sucrose synthase in the potato from the outer membrane to the inner membrane. A simple structure gives a greater surface area, this allows for more contact between sucrose synthase and the amylose and amylopectin, allowing for a faster rate of enzyme activity. Data from the analysis of biologically important macromolecules can be applied to the extraction of biomolecules. This is observed in the extraction of insulin from dogs (Ishida et. al., 1983). This study by Ishida has found a tissue sample in dogs that is abundant in insulin and taken the research further to find how Anastasia affects the biochemical processes in a dog. This research is therefore very useful for the cost efficient extraction of insulin at a high percentage yield.

References

Bj rntorp, P. and Sj str m, L., 1978. Carbohydrate storage in man: speculations and some quantitative considerations. Metabolism, 27(12), pp.1853-1865.

Fredriksson, H., Silverio, J., Andersson, R., Eliasson, A.C. and man, P., 1998. The influence of amylose and amylopectin characteristics on gelatinization and retrogradation properties of different starches. Carbohydrate polymers, 35(3-4), pp.119-134.

Gade, W. and Brown, J.L., 1981. Purification, characterization and possible function of -N-acylamino acid hydrolase from bovine liver. Biochimica et Biophysica Acta (BBA)-Enzymology, 662(1), pp.86-93.

Ishida, T., Lewis, R.M., Hartley, C.J., Entman, M.L. and Field, J.B., 1983. Comparison of hepatic extraction of insulin and glucagon in conscious and anesthetized dogs. Endocrinology, 112(3), pp.1098-1109.

Johansson, L.H. and Borg, L.H., 1988. A spectrophotometric method for determination of catalase activity in small tissue samples. Analytical biochemistry, 174(1), pp.331-336.

Junker, B.H., 2004. Sucrose breakdown in the potato tuber. Mathematisch-Naturwissenschaftliche Fakult t. Universit t Potsdam, 126.

Lang, T., Klein, K., Fischer, J., N ssler, A.K., Neuhaus, P., Hofmann, U., Eichelbaum, M., Schwab, M. and Zanger, U.M., 2001. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics and Genomics, 11(5), pp.399-415.

McCready, R.M. and Hassid, W.Z., 1943. The separation and quantitative estimation of amylose and amylopectin in potato starch. Journal of the American Chemical Society, 65(6), pp.1154-1157.

Kwashiorkor - NHS Choices. nhs.uk. 2016. Website. http://www.nhs.uk/conditions/kwashiorkor/. Accessed 2 Apr. 2017.Smith, R.B., Lougheed, E.C., Franklin, E.W. and McMillan, I., 1979.

The starch iodine test for determining stage of maturation in apples. Canadian journal of plant science, 59(3), pp.725-735.Svegmark, K. and Hermansson, A.M., 1991. Distribution of amylose and amylopectin in potato starch pastes: effects of heating and shearing. Food Structure, 10(2), p.2.

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