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Determine the Gelatine Present in Two Different Samples Using Biuret Protein Assay and Lowry Protein Assay

Introduction to Protein Concentration of G-Globulin

There are variety of protein assays available to scientists. For proteomics studies, protein assays are the essential techniques that are needed to understand. Most of the protein assays can be carried out by two methods: dye-binding assay and copper ion-based protein assay (Thermo Fisher Scientific n.d.). This practical was followed to get knowledge about the principles of common protein estimation assays known as Biuret assay and Lowry assay. In Biuret test, protein samples and Biuret reagent are mixed. Biuret reagent consists of copper ions which combine with the amide groups present in protein sample and yields blue color at 540nm. Protein concentration is quantified by standard graph which shows linear graph among absorbance and the protein concentration (G-Biosciences 2018). This also justifies the statement of Beer-Lambert’s law, which says that the amount of energy emitted or absorbed by a solution is proportional to protein concentration (Edinburgh Instruments n.d.). Biuret test is modified in Lowry assay for more sensitive detection of proteins. In Lowry’s method, initially copper ions are treated with proteins and reduce Cu2+ to Cu+. Further, the copper sulfate solution and proteins react with Folin Ciocalteu reagent, by which amino acids present in the treated samples reduce phosphomolybdate existing in Folin reagent. This yields to the formation of blue color at 700nm. The concentration of protein is identified by plotting a standard curve between the absorbance and standard protein solution, such as that of Bovine Serum Albumin (Khandagle, A n.d).

So, this experiment aims to estimate the protein concentration of g-globulin and gelatine by using Biuret assay and Lowry’s assay.

Reagents

Biuret assay- Copper sulphate- 3g and sodium potassium tartrate- 9g in 500 cm3 of 0.2 mol dm3 sodium hydroxide.

Lowry assay-

  1. 2% (w/v) sodium carbonate in 0.1 mol dm3 NaOH.
  2. 1% (w/v) CuSO4.5H2O
  3. 2% (w/v) potassium sodium tartrate

D: On day of use, 1 vol. of B is mixed with 1 vol. of C and 100 vol. of A to give D.

E: Folin Ciocalteu reagent is diluted 1:3 with distilled water to give E.

Stock solutions of 10mg cm-3 albumin, 5mg gelatin, 5mg cm-3 g-globulin was prepared for Biuret assay. Similarly, for Lowry assay 200μg cm-3 albumin, 100μg cm-3 gelatin, 100μg cm-3 g-globulin was prepared.

Methods of Protein Concentration of G-Globulin

  1. Biuret Assay-

Prepare a series of tubes as follows:

Tube number

Stock solution (cm3)

Water (cm3)

Total volume (cm3)

Protein concentration (μg 0.5 cm-3)

1

0 0

0.5

0.5

0

2

0.1 0

0.4

0.5

1

3

0.2 0

0.3

0.5

2

4

0.3 0

0.2

0.5

3

5

0.4 0

0.1

0.5

4

6

0.5 0

0

0.5

5

7 and 8 (duplicates)

0 0.5 gelatin

0

0.5

2.5

9 and 10 (duplicates)

0 0.5 g-globulin

0

0.5

2.5

Clean test tubes were taken and 2 cm3 of Biuret reagent was added and vortexed for proper mixing of protein and the reagent. All the samples were incubated for at least 15 minutes. Meanwhile, the spectrophotometer was switched on for warm-up and wavelength was adjusted at 540nm. Blank absorbance was set at zero. Likewise, absorbance of each tube was taken and values were recorded to construct the BSA standard curve.

  1. Lowry Assay-

Prepare a series of tubes as follows:

Tube number

Stock solution (cm3)

Water (cm3)

Total volume (cm3)

Protein concentration (μg 0.5 cm-3)

1

0 0

0.5

0.5

0

2

0.2 0

0.4

0.5

20

3

0.2 0

0.3

0.5

40

4

0.3 0

0.2

0.5

60

5

0.4 0

0.1

0.5

80

6

0.5 0

0

0.5

100

7 and 8

0 0.5 gelatin

0

0.5

50

9 and 10

0 0.5 g-globulin

0

0.5

50

In all test tubes, 2.5cm3 of solution D was added and mixed it well and incubated for at least10 minutes. Following incubation, 0.25 cm3 of solution E was added into them with immediate mixing. Further incubated for at least 20 minutes. Blank absorbance was set at zero, and absorbance of each tube was taken at 700nm. All values were recorded to construct the BSA standard curve.

Results of Protein Concentration of G-Globulin

Biuret Protein Assay-

S. No.

Protein concentration (mg/ml)

Absorbance (nm)

1.

0

0

2.

1

0.094

3.

2

0.212

4.

3

0.327

5.

4

0.439

6.

5

0.428

7.

5

0.093

8.

5

0.062

9.

5

0.180

10.

5

0.175

Table 1: Absorbance values obtained by BSA standard protein concentration in Biuret protein assay.

By using values obtained of protein concentration and absorbance mentioned in Table 1, the graph was plotted between the two as seen in Figure 1.

Lowry Protein Assay-

S. No.

Protein concentration (mg/ml)

Absorbance (nm)

1.

0

0

2.

20

0.134

3.

40

0.455

4.

60

0.452

5.

80

0.351

6.

100

0.343

7.

50

0.158

8.

50

0.154

9.

50

0.153

10.

50

0.336

Table 2: Absorbance values obtained by BSA standard protein concentration in Lowry protein assay.

By using values obtained of protein concentration and absorbance mentioned in Table 2, the relation between the two was constructed on the graph as seen in Figure 2.

Results obtained for protein concentration of g-globulin and gelatin is -------by Biuret assay, and ------by Lowry assay respectively. As compare to the known values of protein concentration, the resulting concentration is much higher, which shows that more UV light is absorbed by the proteins.

Discussion on Protein Concentration of G-Globulin

From BSA curves, range of gelatin and g-globulin is---- in case of Biuret assay, and -----in Lowry assay. So, it can be interpreted that the protein concentrations increase linearly with the increase in absorbance (Beer-Lambert’s law). In literature it has been reported that it is a critical step to interpret the curves, as accurate quantification of protein is very sensitive for many clinical applications (Campion et al., 2016). Proteins are made up of various amino acids, like tyrosine, tryptophan, leucine, arginine etc. So, tyrosine and tryptophan acts as the building blocks used to construct proteins. These amino acids absorb UV light in spectrophotometer. The higher the value of amino acids in the solution, more UV light is absorbed by the protein. Gelatin derives from collagen and 100g of gelatin contains 1g of tryptophan and 0g of tyrosine; whereas 100g of g-globulin contains 6.8g of tryptophan and 2.9g of tyrosine (Chang and Zhang, 2017). So, we infer from this that the sample containing gelatin absorbs less light, and the one which contains g-globulin absorbs more light.

One major real-time discrepancy occur in this experiment is of limited range of absorbance in the BSA curve. More protein samples should have been prepared, so that we have wider range of absorbance values. It would be easy to extrapolate the protein concentration value corresponding to absorbance. Another limitation is that the protein concentrations of gelatin and g-globulin obtained by Biuret assay and Lowry assay shows a wide range of difference. For Biuret assay, protein concentration is very less as compare to Lowry assay. In order to overcome such discrepancies, it is suggested to repeat the experiment. Future studies should aim to resolve these limitations before working on this experiment.

References for Protein Concentration of G-Globulin

Campion, EM, Loughran, ST & Walls, D 2016, Protein quantitation and analysis of purity. Clifton, NJ (eds)Protein Chromatography: Methods and Protocols, Switzerland AG, pp. 225-255. https://doi.org/10.1007/978-1-4939-6412-3_12

Chang, SKC & Zhang, Y 2017, Protein Analysis. Nielsen, S (eds)Food Analysis, Food Science Text Series, Springer, Cham. https://doi.org/10.1007/978-3-319-45776-5_18

Edinburgh Instruments n.d., The Beer-Lambert’s law, Available at: https://www.edinst.com/blog/the-beer-lambert-law/#:~:text=The%20Beer%2DLambert%20law%20states,calculated%20by%20measuring%20its%20absorbance.

G-Biosciences (2018), Biuret protein assay, Available at: https://www.gbiosciences.com/image/pdfs/protocol/402B_Biuret_Protein_Assay.pdf

Khandagle, A n.d., Total proteins estimation by Lowry method, Available at: https://www.slideshare.net/AbhayKhandagle/lowry-method-for-protein-estimation

Thermo Fisher Scientific n.d., Chemistry of protein assays, Available at: https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemistry-protein-assays.html

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