Experiment 9: Sugar Analysis
Performed: April 9, 2014 Due: April 18, 2014
Writer: Naomi Rodman Reviewer: Robert Burnham
Unknown 11
“I have neither given nor received unauthorized assistance on this assignment”
Objective: Determining the glucose and mannose concentration of an unknown solution by finding the total carbohydrate concentration via a BCA assay and the glucose concentration using a hexokinase (HK) coupled reaction.
Results (BCA Sugar Assay; Glucose + Mannose Analysis):
The components, molecular weights and the mass used to create the BCA reagent are presented in Table 1. Mixtures of solutions A and B, denoted in parenthesis, were created then equally mixed to create the BCA working reagent used in both the standard curve and unknown assay.
Table 1: Composition of BCA Reagent
Reagent
Molecular Weight (g/mol)
Mass In Solution (g)
BCA (A)
388.2
0.1924
Na2CO3H2O (A)
124.0
6.35
NaHCO3 (A)
84.0
2.42
CuSO4*5H2O (B)
249.7
0.1248
L-Serine (B)
105.1
0.1262
A 100 mL solution of 5 mM glucose (180.2 g/mol) was diluted 1:10, 0.4 mL glucose with 3.6 mL H2O, was used to create the standard curve. The components and volumes used to create the glucose standard curve are shown in Table 2.
Table 2: Glucose Standard Curve
Glucose (nmol)
Dilution Factor
Diluted Glucose (µL)
BCA (µL)
H2O (µL)
5
10
10
500
490
10
10
20
500
480
15
10
30
500
470
20
10
40
500
460
25
10
50
500
450
The unknown 11 curve was measured similarly: the solution was diluted 1:100 and the same volumes of the three components (unknown solution instead of diluted glucose) were added as in the standard. After incubation in 100°C for 15 minutes and a 20 minute cool down period, each standard and unknown solution was assayed at 562 nm. Some assay solutions were repeated for greater accuracy and the final results used are displayed in Table 3.
Table 3: Results of BCA Assay
Standard Curve (nmoles)
Standard Absorbance
Unknown (µL)
Dilution Factor for Unknown
Unknown Absorbance
5
0.3548
10
100
0.0940
10
0.7433
20
100
0.3388
15
1.2212
30
100
0.6987
20
1.6579
40
100
1.2859
25
1.2011
50
100
1.1140
The standard curve is shown in Figure 1 and only the points on the regression line were used to create the equation since at 25 nmoles the absorbance was non-linear and an outlier.
The concentration of glucose and mannose in the unknown was 28788.6 nmole/mL (28.788 mM, 5.1875 g/L) using the unknown absorbance value 0.6987 and 0.03 mL assay volume with the calculation shown below. y = 0.0809x x = = 8.6365 nmole of protein = 287.88 nmole/mL of protein glucose in the diluted solution
287.88 nmole /mL * (dilution factor) = 287.88 * 100 = 28788.6 nmole/mL = 28.788 mM of glucose + mannose in the original solution (Glucose Analysis):
The addition of HK in an assay only measured the glucose in solution. To find the concentration of glucose, the rate and absorbance of a standard curve and unknown 11 taken at 562 nm are portrayed in Tables 4 and 5.
Table 4: Standard Curve
Nmoles
Dilution Factor
Volume in assay (µL)
Absorbance
Rate (ΔA/min)
25
10
50
0.2054
0.1540
50
10
100
0.4831
0.3167
100
1
20
0.3885
0.2641
150
1
30
0.5529
0.3023
200
1
40
0.7556
0.4030
250
1
50
0.9778
0.4516
The linear regression equation shown in Figure 2 was calculated using all the values but 50 nmoles from Table 4.
The absorbance values for the unknown are shown in Table 5.
Table 5: Unknown 11 Measurements
Nmoles
Dilution Factor
Volume in assay (µL)
Absorbance
Rate (ΔA/min)
25
1
5
0.15949
0.1313
50
1
10
0.1290
0.2141
100
1
20
0.2758
0.3899
150
1
30
0.7184
0.5838
200
1
40
1.2702
0.5430
250
1
50
1.6908
0.5208
The graph of the standard curve from the absorbance values is shown in Figure 2. The outlier, 50 nmole, is shown in blue and not used in the equation.
The concentration of glucose (MW 180.2) in the unknown using the absorbance value 0.7184 at 0.03 mL was calculated as before and found to be 6140.2 nmole/mL (6.1402 mM, 1.1064 g/L).
Discussion:
The BCA assay measures the concentration of
