Name: Gladys M.

Quiatchon Section/Group: 1L/Group3

Date Performed: Dec. 5, 2011 Date Submitted: Dec. 12, 2011

EXERCISE 4 ESTIMATION OF PROTEIN CONCENTRATION BY SPECTROPHOTOMETRY

I.

RESULTS AND DISCUSSION

To estimate the protein concentrations in the egg albumin isolate, the Biuret method was used in the experiment. Biuret method is said to be the simplest method in the estimation of protein concentration. It is sensitive to the amino acid composition of the protein. Based on the manual, the reagent used in this method is composed of 0.09375g of CuSO4.5H2O and 0.375g of sodium potassium tartrate (NaKC4H4O.6H2O), dissolved in 31.25mL of water, and mixed with 18.75mL of 10% NaOH which is subsequently diluted to 250mL with water. Basically, this test produces violet or purple color when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. The color intensifies as the number of peptide bonds and the protein concentration increases. The amount of the charge transfer absorption bond is linearly proportional to the mass of protein present in the solution. This method is different to other tests in a way that there is no interference from other substances that adsorb at lower wavelengths. Also, this technique is less sensitive to protein type. It may be applied to any proteins, not just specific side groups. Other methods, aside from the Biuret method (BI), can be used in the protein determination. These include the Lowry method (LO) and the Warburg-Christian method (WC). With comparison to the Biuret method, the Lowry method combines the Biuret reagent with another reagent (Folin-Ciocalteaue phenol reagent) which reacts with tryptophan and tyrosine. This gives a bluish color which can be read in 500-750nm. Also, LO is more sensitive to low concentrations of proteins than the Biuret method. On the other hand, the Warburg-Christian method has a lower range of absorbance of 260-280nm than Biuret method (540nm and above). The only advantage of WC is that it is more time and material efficient. However, its disadvantage is the same with that of BI. The presence of UV-absorbing compounds may interfere and cause errors in the data. Table 1. Absorbance of standard BSA solutions. Test tube no. 1 2 3 4 5 6 BSA concentration (mg/mL) 0 2 4 6 8 10 A540 0.000 0.050 0.112 0.152 0.218 0.281

Standard bovine serum albumin (BSA) with increasing concentrations was used as basis for the determination of protein concentration of the unknown sample. Based on the values obtained, the standard curve in Figure 1 was constructed. This curve serves as the illustration of the derivation of the actual protein concentration of the unknown sample. Using Linear Regression (LR), certain values were known. These include r or the correlation coefficient which is 0.9981 which tells that there is a very strong positive linear relationship between the concentration and absorbance; the A or y intercept is -3.7143 x 10-3 which means that the curve started increasing at this point; the B or slope is 0.0278 which indicates that there was increase of 0.0278 in absorbance. The standard curve formed is in line with the Beers law whereas the graph must originate from (0 concentration, 0 absorbance) and constantly increase with every increase in concentration.
0.3

r = 0.9981
0.25 Absorbance at 540nm

A (y intercept) = -3.7143 x 10-3 B (slope) = 0.0278

0.2

0.15

0.1

0.05

0 0 2 4 6 Concentrattion of BSA stock 8 10

Figure 1. Standard curve for protein concentration determination. According to the Beers law, the absorbance (540nm) is directly proportional to the concentration of a solution, in the case of the experiment, the BSA stock. Moreover, Beers law is limited only to dilute solutions. This is because it is only applicable for monochromatic radiation and the nature of absorbing materials is fixed over the concentration range. The bovine serum albumin or BSA was used as the standard for protein content determination because it is an appropriate protein standard. It is an ideal one to use since its theoretical protein concentration is suitable into the range where the Biuret method is useful. Other proteins can be used instead. These include egg white lysozyme. But this can only be done in this particular assay since choosing the appropriate standard may also be based on what method to use, the protein concentration and more.

Table 2. Absorbance of crude egg albumin samples. Test tube no. 7 8 9 10 11 BSA concentration (mg/mL) Undiluted 1:2 1:3 1:7 1:10 A540 0.544 0.550 0.548 0.426 0.216

Other absorbances were known using the Biuret assay and are indicated in Table 2. And based on the standard curve formed on BSA, the protein content of crude egg albumin was computed and results are shown in Table 3. Table 3. Calculated values for protein content of crude egg albumin.

Protein dilution Parameter 1:10 Dilution factor Diluted Protein concentration (mg/mL) Actual Protein concentration (mg/mL) Protein %(w/w) in egg albumin 11 7. 8912 86.8035 72.34%

II.

SAMPLE CALCULATIONS

1. For standard curve Given: Stock Solution = 20mg/mL = 0.5M NaOH 0-10 mg BSA with 1.0mL volume

Volume BSA stock Use: C1V1 = C2V2

Volume BSA stock = (C2V2) / C1 = (0 x 1.0) / (20) = 0 mL y Volume of NaOH = 1.0 mL Volume of BSA stock = 1.0 mL 0 mL = 1.0 mL

Test tube 1 = 0 mL BSA stock + 1.0 mL NaOH Test tube 2 = 0.1 mL BSA stock + 0.9 mL NaOH Test tube 3 = 0.2 mL BSA stock + 0.8 mL NaOH Test tube 4 = 0.3 mL BSA stock + 0.7 mL NaOH Test tube 5 = 0.4 mL BSA stock + 0.6 mL NaOH Test tube 6 = 0.5 mL BSA stock + 0.5 mL NaOH

2. Calculation of values for protein content of crude egg albumin (Table 3) y Diluted Protein concentration = absorbance (x) = 0.216 (x) = 7.8912 mg/mL y Actual Protein concentration = Dilution factor x Diluted protein concentration = (11) (7.8912 mg/mL) = 86.8035 mg/mL Protein %(w/w) in egg albumin = Actual Protein Concentration / BSA Stock concentration = [(86.8035 mg/mL) / (120 mg/mL)] x 100 = 72.34% (w/w) or 72.34g protein per 100g of isolate

Resources: http://spinner.cofc.edu/genchemlab/beers.htm http://people.umass.edu/~mcclemen/581Proteins.html http://btprotocols-maulik.blogspot.com/2007/04/to-perform-estimation-of-protein-by.html