The Estimation of Protein Molecular Weight Through Gel Filtration Chromatography

Experimental Data & Explanation

Table 1: Calibration of Sephadex G-75 Column

Fraction #: Start Vol (mL): End Vol (mL): Midpoint Vol: ABS400: ABS620: Blue-D (µg/mL): DNPAA (µg/mL): Blue-D (µg): DNPAA (µg):

1 0 1 0.5 0 0 0.0 0.0 0.0 0.0

2 1 2 1.5 0 0 0.0 0.0 0.0 0.0

3 2 3 2.5 0 0 0.0 0.0 0.0 0.0

4 3 4 3.5 0 0 0.0 0.0 0.0 0.0

5 4 5 4.5 0 0.002 2.3 0.0 2.3 0.0

6 5 6 5.5 0.471 1.003 1139.8 28.3 1139.8 28.3

7 6 7 6.5 1.23 2.618 2975.0 73.9 2975.0 73.9

8 7 8 7.5 0.371 0.794 902.3 22.3 902.3 22.3

9 8 9 8.5 0.151 0.327 371.6 9.1 371.6 9.1

10 9 10 9.5 0.092 0.201 228.4 5.5 228.4 5.5

11 10 11 10.5 0.004 0.139 158.0 0.2 158.0 0.2

12 11 12 11.5 0.041 0.687 780.7 2.5 780.7 2.5

13 12 13 12.5 0.023 0.053 60.2 1.4 60.2 1.4

14 13 14 13.5 0.014 0.033 37.5 0.8 37.5 0.8

15 14 15 14.5 0.013 0.027 30.7 0.8 30.7 0.8

16 15 16 15.5 0.024 0.018 20.5 1.4 20.5 1.4

17 16 17 16.5 0.141 0.011 12.5 8.5 12.5 8.5

18 17 18 17.5 0.746 0.007 8.0 44.8 8.0 44.8

19 18 19 18.5 2.795 0.005 5.7 168.0 5.7 168.0

20 (1:4) 19 20 19.5 1.749 0.001 1.1 105.1 1.1 105.1

21 (1:6) 20 21 20.5 2.588 0 0.0 155.5 0.0 155.5

22 (1:8) 21 22 21.5 2.358 0.005 5.7 141.7 5.7 141.7

23 (1:6) 22 23 22.5 2.893 0.003 3.4 173.9 3.4 173.9

24 (1:4) 23 24 23.5 2.884 0 0.0 173.3 0.0 173.3

25 (1:4) 24 25 24.5 1.611 0.003 3.4 96.8 3.4 96.8

26 (1:2) 25 26 25.5 1.467 0.004 4.5 88.2 4.5 88.2

27 26 27 26.5 1.642 0 0.0 98.7 0.0 98.7

28 27 28 27.5 0.944 0 0.0 56.7 0.0 56.7

29 28 29 28.5 0.596 0.004 4.5 35.8 4.5 35.8

30 29 30 29.5 0.389 0 0.0 23.4 0.0 23.4

31 30 31 30.5 0.208 0 0.0 12.5 0.0 12.5

32 31 32 31.5 0.185 0 0.0 11.1 0.0 11.1

33 32 33 32.5 0.139 0 0.0 8.4 0.0 8.4

34 33 34 33.5 0.085 0 0.0 5.1 0.0 5.1

35 34 35 34.5 0.059 0 0.0 3.5 0.0 3.5

36 35 36 35.5 0.041 0 0.0 2.5 0.0 2.5

37 36 37 36.5 0.039 0 0.0 2.3 0.0 2.3

38 37 38 37.5 0 0 0.0 0.0 0.0 0.0

39 38 39 38.5 0 0 0.0 0.0 0.0 0.0

Table 1 displays the results of the calibration of the columns. Based off the results, the Blue-D absorbed better at 620 nm, while the DNPAA absorbed more at 400 nm. Furthermore, the data can be graphed and the Vt and Vo can be determined.

By using the data from Table 1, Figure 1 was able to be created. By convention, Vt and Vo are taken at the volume with the maximum peak. In this case, Vt is no the highest peak on the graph, but would be if not diluted. Based on the graph, the Vo = 7 mL and Vt = 22 mL. Using these data points, the Vi can be determined to be 15 mL.

Calculations:

Beer’s Law Concentration: Conc. = (Abs./ɛ·l)

EX: X = (0.471/0.01664 µg/mL-1 cm-1) · (1 cm)

X = 28.3 µg/mL

Table 2: Gel Filtration Seperation of BSA and Lysozyme

Fraction #: Start Vol. (mL): End Vol. (mL): Midpoint Vol. : ABS280: Conc. BSA (mg/mL): Conc. Lysoz (mg/mL): Amnt. Of BSA (mg): Amnt. Of Lysoz (mg):

1 0 1 0.5 0 0.00 0.00 0.00 0.00

2 1 2 1.5 0 0.00 0.00 0.00 0.00

3 2 3 2.5 0 0.00 0.00 0.00 0.00

4 3 4 3.5 0.004 0.01 0.00 0.01 0.00

5 4 5 4.5 0.007 0.01 0.00 0.01 0.00

6 5 6 5.5 0.02 0.03 0.00 0.03 0.00

7 6 7 6.5 0.35 0.54 0.00 0.54 0.00

8 7 8 7.5 0.412 0.64 0.00 0.64 0.00

9 8 9 8.5 0.266 0.41 0.00 0.41 0.00

10 9 10 9.5 0.173 0.27 0.00 0.27 0.00

11 10 11 10.5 0.163 0.25 0.00 0.25 0.00

12 11 12 11.5 0.174 0.00 0.27 0.00 0.27

13 12 13 12.5 0.247 0.00 0.38 0.00 0.38

14 13 14 13.5 0.431 0.00 0.67 0.00 0.67

15 14 15 14.5 0.65 0.00 1.01 0.00 1.01

16 15 16 15.5 0.803 0.00 1.24 0.00 1.24

17 16 17 16.5 0.83 0.00 1.29 0.00 1.29

18 17 18 17.5 0.751 0.00 1.16 0.00 1.16

19 18 19 18.5 0.608 0.00 0.94 0.00 0.94

20 19 20 19.5 0.449 0.00 0.70 0.00 0.70

21 20 21 20.5 0.314 0.00 0.49 0.00 0.49

22 21 22 21.5 0.212 0.00 0.33 0.00 0.33

23 22 23 22.5 0.188 0.00 0.29 0.00 0.29

24 23 24 23.5 0.09 0.00 0.14 0.00 0.14

25 24 25 24.5 0.059 0.00 0.09 0.00 0.09

26 25 26 25.5 0.039 0.00 0.06 0.00 0.06

27 26 27 26.5 0.027 0.00 0.04 0.00 0.04

28 27 28 27.5 0.011 0.00 0.02 0.00 0.02

29 28 29 28.5 0.002 0.00 0.00 0.00 0.00

30 29 30 29.5 0 0.00 0.00 0.00 0.00

Total (mg) N/A N/A N/A N/A N/A N/A 2.16 9.12

%Recov N/A N/A N/A N/A N/A N/A 180.19 759.89

Table 2 displays the results of the gel filtration for separation of two components. Based on the results, the heavier protein was filtered out first and then the lighter one. This is verified by the absorbance readings seen in the table. Furthermore, the percent recovery is incorrect because it is above 100%. This error could have been caused by inaccurate absorbance readings.

Figure 2 is the graphical representation of the data seen in Table 2. Based on the graph, the Ve for BSA = 8 mL and Ve for Lysozyme = 17 mL. These values can be verified by the table above where these volumes have the highest absorbance readings.

Calculations:

Beer’s Law Concentration: Conc. = Abs · 1.55

EX: X = (0.412) · 1.55

X = 0.64 mg/mL

Percent Recovery: Amt. Obtained/Original Amt · 100

EX: X = 2.16 mg/1.2 mg · 100 = 180%

Got 1.2 mg from 4 mg/mL · 0.3 mL = 1.2

Table 3: Standard Curve Data

Vo (mL): Vt (mL): Ve (mL): Kav: MW (kD): Resolution:

BSA 7 22 8 0.0667 67 0.72

Lysozyme 7 22 17 0.667 14.4

Table 3 shows the results of calculating the resolution and the data to make a standard curve for determining other proteins weight. Based on the resolution, the proteins did not fully separate because the value is not of 1. However for complete separation, the resolution would be greater than 1.5. Furthermore, by using Figure 2, the distance and the widths need for the resolution can be determined.

Figure 3 is the standard curve from the data in Table 3. By using this graph, the molecular weight of unknown proteins can be estimated by using the line of best fit and/or the linear regression equation. The Kav represents the fraction of stationary phase available for diffusion of a given solute species.

Calculations:

Kav: Ve – Vo / Vt – Vo

EX: X = 8 mL – 7 mL / 22 mL – 7 mL = 0.0667

Resolution: 2D/W1 + W2

EX: X = 2(17-8) / 7 + 18 = 0.72

Discussion of Experiment/Errors & Reflection:

Gel filtration chromatography is just another form of chromatography, and is typically used to separate mixtures with high molecular weight biomolecules. The purpose of the this lab is to practice gel filtration chromatography and understand the applications for which they are used for. More specifically, the laboratory activities performed to accomplish the purpose are: Calibrate a Sephadex G-75 column, use that column to separate components of a known protein mixture and estimate the recovery of materials applied to the gel filtration columns.

In gel filtration chromatography, the stationary phase is a porous gelatin - like matrix in the form of beads held in columns. Each type of gel filtration column has unique characteristics needed to understand and predict the separation of the components in mixtures; the total column volume (Vt), the interstitial volume (Vi), and the void volume (Vo). The volumes can be determined by a process called column calibration. Table 1 and Figure 1 display the results of this process. Once calibrated and these values are determined, the column and the values can be used to estimate the molecular weight of components in other sample mixtures. In this case, based on the results, the Vo was determined by the Blue dextran data; more specifically the tip of the peak on Figure 1. This was like wise for Vt, where DNPAA was used because of its lower molecular weight.

Like previously stated, gel filtration chromatography is used to separate components of mixtures. Table 2 and Figure 3 show the results of separation of two proteins via Sephadex ge filtration. Based on how the order of elution goes from a gel filtration column, the BSA because of its higher molecular weight is the first part (hill) in Figure 2. Figure 2 was created by using the absorbance readings and comparing them the elution volumes. From Figure 2 the Ve a volume, which is the volume of mobile phase needed to carry a group of similar weighted molecules through the column, and can be determined by the peaks of each “hill”. These values, in conjugation with Vt and Vo, help calculate the K¬av values. Furthermore, Figure 2 displays the widths of each peak and the distance between each Ve that is needed to determine the resolution. In addition to all that, the percent recovery is displayed in Table 2. Inaccuracy is involved here with the percentages being so high. Typically, machines are not blame for a source of error, but with the past problems with the spectrophotometer having inaccuracy; this could possibly be the most probable cause.

In Table 3, the Kav and the resolution values are displayed. The resolution value relates to how completely separate the two proteins are. Typically, the resolution value is around 1 – 1.5 meaning 98% to 99.8% has completely separated. Because the value seen in the table is much lower meaning there was not complete separation of the proteins…leading to the data being in accurate. In addition, the data in Table 3 was used to the standard curve seen in Figure 3. Figure 3 is an example of a Kav vs. log of molecular weight, and by using the graph, other proteins can help be identified. The Kav value is the fraction of the stationary phase in gel filtration chromatography that is available for diffusion of a given solute.

In any experiment, there is the possibility of errors occurring that can alter the results. During the separation portion of the lab, some sort of error occurred that led to the percent recovery to be over 100%. The spectrophotometer could possibly be the cause of that, but because the resolution value is lower, then a problem occurred in the separation of the proteins that led to inaccuracy in the data. Furthermore, if the fluid ever fell below the surface, then cracking of the gel will occur. If this happens, this leads to inaccurate results. Obviously, there is more cause of error that could have occurred, but these are the biggest factors.