Manning, Michael
11/21/14
Tuesday AM
TA: Rebecca Johnson
I. Purpose/Objective:
The purpose of this lab was to analyze the functional properties in proteins with particular
interest on solubility by adjusting the pH of different solutions and using the Bradford
Assay to make a standard curve.
II. Introduction:
In this experiment we used a Bradford Assay to set of a standard curve for protein
concentration. We did this by using known concentrations of BSA (Bovine Serum
Albumin), which we then used to find concentrations of whey and casein at different
pH’s and temperatures.
Having an understanding of protein functionality and solubility is important for the food
industry so that everything from processing to packaging can be better understood to
make higher quality products (depending on food or longevity). Protein Solubility is
particularly important for foam making and emulsification.
III. Procedure:
The procedure followed for the experiment is found in “Principles of Food Composition,
Laboratory Manuel, FS&T 101A” (2014) Lab 4, Protein Functionality: Solubility and
Foam Formation, pages 39-49.
IV. Data/Graphs:
Table 1A. Concentration and absorbance data for standard curve made with BSA for
Whey Sample.
Standard
0.0
0.1
0.2
0.4
0.8
mg/mL
Absorbance 0.000 0.110 0.269 0.498 0.960
@595nm
Table 1B. Concentration and absorbance data for standard curve made with BSA for
Casein Sample.
Standard mg/mL
0.0
0.1
0.2
0.4
0.8
Absorbance @
0.000
0.069 0.161 0.279 0.422
595nm
Table 2A. Absorbance data reflecting the solubility of heated and non-heated whey
proteins in the designated pH environments
pH
2.68
3.65
4.62
5.65 6.41
7.54 8.52
Absorb Heat Treated
0.368 0.480 0.244 0.438 0.676 0.658 0.510
Non-Heat
0.376 0.438 0.408 0.470 0.626 0.584 0.432
Treated
Table 2B. Absorbance data reflecting the solubility of heated and non-heated casein
proteins in the designated pH environments.
pH
2.55 3.42
4.60
5.67 6.60
7.64
8.51
Absorb Heat Treated
0.270 0.268 0.350 0.289 0.267 0.323 0.263
Non-Heat
0.304 0.246 0.250 0.210 0.279 0.307 0.250
Treated
Table 3A. Calculated data showing percent solubility, heated or non-heated, as a function
of pH for Whey.
Protein
pH
% Solubility (Heat) % Solubility (No
Heat)
Whey
2.68
30.31
30.97
3.65
39.54
36.08
4.62
20.10
33.61
5.65
36.08
38.71
6.41
55.68
51.56
7.54
54.20
48.10
8.52
42.01
35.58
Table 3B. Calculated data showing percent solubility, heated or non-heated, as a function
of pH for Casein.
Protein
pH
% Solubility (Heat) % Solubility (No
Heat)
Casein
2.55
23.50
26.46
3.42
23.32
21.41
4.60
30.46
21.76
5.67
25.15
18.28
6.60
23.24
24.28
7.64
28.11
26.72
8.51
22.89
21.76
Absorbance @ (595nm)
Standard Curve - Whey
1.200
y = 1.2141x
R² = 0.9981
1.000
0.800
0.600
Abs
0.400
Linear (Abs)
0.200
0.000
0.0
0.5
[BSA] mg/mL
1.0
Figure 1A: Graph of the Bradford Assay standard curve for Whey. Five known
concentrations of bovine serum albumin were used. Absorbance readings obtained from a
Spec 20D spectrophotometer.
Absorbance @ (595nm)
Standard Curve - Casein
0.500
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
y = 0.5745x
R² = 0.9462
Abs
Linear (Abs)
0.0
0.2
0.4
0.6
[BSA] mg/mL
0.8
1.0
Figure 1B: Graph of the Bradford Assay standard curve for Casein. Five known
concentrations of bovine serum albumin were used. Absorbance readings obtained from a
Spec 20D spectrophotometer.
% Solubility as a fx of pH - Whey
70.00
% Solubility
60.00
50.00
40.00
% Solubility Heated
30.00
% Solubility NonHeated
20.00
10.00
0.00
0.00
5.00
pH
10.00
Figure 2: Graph of percent solubility of heated and non-heated whey protein as a function
of pH. Percent solubility calculated from best-fit line of Bradford Assay standard curve
for protein concentration against the original whey protein concentration of 0.1/100mL.
% Solubility as a fx of pH - Casein
35.00
% Solubility
30.00
25.00
20.00
% Solubilty Heated
15.00
% Solubility NonHeated
10.00
5.00
0.00
0.00
5.00
pH
10.00
Figure 3: Graph of percent solubility of heated and non-heated casein protein as a
function of pH. Percent solubility calculated from best-fit line of Bradford Assay
standard curve for protein concentration against the original casein protein concentration
of 0.05g/100mL.
V. Calculations:
y = absorbance (Abs), x = concentration of unknown (whey)
1. Final [Whey]:
y = 1.2141*x
@ pH 5.65  Heat Treated Abs: 0.438
0.438/1.2141 = x = 0.3608 mg/mL
2. % Solubility of Whey:
(0.3608 mg/mL) * (1.00mg/mL) * 100 = 36.08% Solubility of Whey
3. Final [Casein]:
y = 0.5745*x
@pH 5.67  Heat Treated Abs: 0.289
0.289/0.5745 = x = 0.5030 mg/mL
4. % Solubility of Casein:
(0.5030 mg/mL) * (0.5mg/mL) * 100 = 26.50% Solubility of Casein
VI. Discussion:
The concentrations of whey and casein were determined by making a standard curve. In
order to make a standard curve we has to obtain five different samples of BSA, which has
similar properties as whey and casein. In figure 1A and 1B, the best-fit line equation was
calculated and using this best-fit equation we were able to use it, in conjunction with the
absorbance readings, to calculate the concentration of the proteins. Potential errors may
have been from reading the absorbance incorrectly (which would explain higher and
lower values) or fingerprints on the glass tubes causing the absorbance to be read
incorrectly (which would point to higher readings).
The isoelectric point of a protein is where the protein has no net charge. It is at this point
that the protein is least soluble because the polarity has diminished, decreasing its ability
to act with the water and is at increased risk for falling out of solution. Looking at both
figure 2 and 3, the isoelectric point varied throughout different pHs for both heat treated
and non-heat treated solutions in both whey and casein. There was a marked lull at pH
4.60 for heat-treated for whey indicating was the most significant point for whey to be at
its isoelectric point. For the non-heat treated, there was no significant interaction with the
water resulting in a poor isoelectric point. Looking at the casein % solubility, the
isoelectric point for casein was 18.28% at a pH of 5.67. The low solubility of this protein
indicates that casein is more sensitive to pH and showing that is has a weaker interaction
with water making it a less stable protein.
Percent solubility for Whey and Casein were calculated using the Bradford Assay with
the absorbance values obtained in class. There is a direct correlation between a higher
absorbance reading and a higher protein concentration; the higher the absorbance reading,
the more protein there was in solution, which means higher protein solubility. The
contrast is also true that the lower the protein concentration the lower the percent
solubility which therefore leads us to discern that the lowest point of % solubility also
had the lowest amounts of protein concentration. At the isoelectric point, the percent
solubility was the lowest because they protein had precipitated out of solution. Whey
proved to be heat sensitive but stable at varying pHs but casein was the opposite. Casein
was heat stable and pH sensitive.
VII. Conclusion:
Different functional properties of casein and whey were observed adjusting for heat
treatment, no heat treatment and varying pHs. The results were that the casein was heat
stable and whey was not but whey was pH stable and casein was not. The Bradford Assay
method served to be useful since the BSA acts functionally very similar to casein and
whey.
VIII. Questions:
1. For Whey the least stable point was at a pH of 4.62 for both heat-treated and non-heat
treated. Casein was different for both heat-treated and non-heat treated. Heat-treated was
at pH 5.67 and non-heat treated was a 6.60. The protein is at it’s isoelectric point when it
is least soluble and this means the protein has no charge and cannot interact with water
which leads to the protein precipitating out of solution.
2. The lowest % solubility for heat-treated was 20.10% for whey and 23.24% for casein.
These results were not to be expected. Casein would normally be much lower because of
its micellular molecular structure. The casein micelles should have precipitated more at
the pH. When GMP is neutralized the structure of the casein micelles become
hydrophobic and do not interact with water. The percent solubility for non-heat-treated
for whey was 33.61% and 18.28% for casein. The whey is more stable without heat
treatment because whey is more stable at low heat and is has a stable pH. The lower
solubility of the whey at high heat was due to the disulfide bridges rearranging from
structural modification. Exposing hydrophobic parts unable to react with water.