Aaron Vo 11/4/15 A03 Wednesday 2-5 PM Group 7/HS LAB 6- Discovering how ph/heat Change Affects Solubility I. PURPOSE/OBJECTIVE: The purpose of lab 6 was to observe how altering the ph or heat to proteins (whey and casein) would affect their solubility. To do this, students created separate standard curves using the Bradford Assay for each protein. Then, using the line-ofbest-fit, students could solve for the protein concentrations and ultimately the % solubility. II. INTRODUCTION: The physical and chemical properties of proteins are what determine the behavior or usefulness of them. In other words, shape determines function. For example, a polar functional group on an amino acid chain would increase its net charge and change its solubility. Furthermore, the function of a protein is dependent on whether the protein is in its natural form or if it is denatured. Proteins can become denatured in the presence of stimuli such as ph or heat change. As we will see in this lab, denaturation may also affect the solubility of a protein. Solubility of proteins is important because it is the backbone of other functions such as viscosity, emulsification, and foaming. Generally, solubility increases with increased polarity and decreases with increases molecular weight. To elaborate further, a soluble protein must have hydrophilic groups like a hydroxyl, amino, or carboxyl that interact with the waters surface. The properties of proteins may seem like a trivial thing to the everyday consumer, but it is vital for food scientists to be able to understand them. Knowing these properties means that one could manipulate their foods texture or taste based on the inclusion of certain proteins. It also plays an important role in processing and storage.
III. PROCEDURE: The procedures for lab 6 can be found in the FST 101A- Food Chemistry Laboratory Fall 2015 manual on pages 52-57. There were no modifications to this lab. IV. DATA/RESULTS: Table 1: Table for concentration and absorbance values for my standard curve (whey). Tube Type I H2O 1 mg/ml [BSA] Avg. Standard Absorbance (ml) BSA (ml) (mg/ml) Absorbance Deviation 1 0.100 0.000 0.000 0.000 0.000 0.000 2 0.090 0.010 0.100 0.223 3 0.090 0.010 0.100 0.243 0.233 0.01414 4 0.080 0.020 0.200 0.260 5 0.080 0.020 0.200 0.354 0.307 0.06647 6 0.060 0.040 0.400 0.536 7 0.060 0.040 0.400 0.538 0.537 0.00141 8 0.020 0.080 0.800 0.880 9 0.020 0.080 0.800 1.050 0.965 0.12021
My Bradford Assay Standard Curve (Whey) Absorbance at 595 nm 1.200 1.000 0.800 0.600 0.400 0.200 y = 1.0635x + 0.1117 R² = 0.99836 0.000 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 [BSA] (mg/ml) Figure 1: Graph of whey standard curve. Has line of best fit, r 2 value, and error bars. Table 2: Table for concentration and absorbance values for the other group s standard curve (casein). Tube Type I H2O (ml) 1 mg/ml BSA (ml) [BSA] (mg/ml) Absorbance Avg. Absorbance Standard Deviation 1 0.100 0.000 0.000 0.000 0 0 2 0.090 0.010 0.100 0.170 3 0.090 0.010 0.100 0.168 4 0.080 0.020 0.200 0.300 5 0.080 0.020 0.200 0.243 6 0.060 0.040 0.400 0.562 7 0.060 0.040 0.400 0.520 8 0.020 0.080 0.800 0.985 9 0.020 0.080 0.800 1.030 0.169 0.00141 0.272 0.0403 0.541 0.02970 1.008 0.03182
Other Group's Bradford Assay Standard Curve (Casein) Absorbance at 595 nm 1.200 1.000 0.800 0.600 0.400 0.200 y = 1.2099x + 0.0438 R² = 0.99906 0.000 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 [BSA](mg/mL) Figure 2: Graph of casein standard curve. Has line of best fit, r 2 value, and error bars. Table 3: Table of absorbance data for whey at the various actual ph s. Absorbance (unheated) Absorbance (heated) ph 0.392 0.351 2.50 0.400 0.406 3.60 0.345 0.279 4.50 0.398 0.410 5.50 0.350 0.448 6.50 0.366 0.369 7.50 0.552 0.620 8.60 Table 4: Table of absorbance data for casein at the various actual ph s. Absorbance (unheated) Absorbance (heated) ph 0.462 0.448 2.70 0.085 0.072 3.65 0.470 0.388 4.70 0.544 0.428 5.32 0.516 0.476 6.70 0.560 0.548 7.50 0.560 0.566 8.35
Table 5: Table of % solubility of unheated/heated whey at various ph s. ph % Solubility of Unheated Whey % Solubility of Heated Whey 2.50 35.1418273 30.00156715 3.60 36.14480489 36.89703808 4.50 29.24933396 20.97476885 5.50 35.89406049 37.39852688 6.50 29.87619495 42.16267043 7.50 31.88215013 32.25826673 8.60 55.20137909 63.72668861 70 60 Overlay of % Solubility for Unheated/ Heated Whey % Solubility 50 40 30 20 % Solubility of Unheated Whey % Solubility of Heated Whey 10 0 0.00 2.00 4.00 6.00 8.00 10.00 ph Figure 3: Overlay % solubility of unheated/heated whey at various ph s.
Table 6: Table of % solubility of unheated/heated casein at various ph s. ph % Solubility of Unheated Casein % Solubility of Heated Casein 2.70 69.12968014 66.81543929 3.65 6.810480205 4.661542276 4.70 70.45210348 56.89726424 5.32 82.68451938 63.50938094 6.70 78.05603769 71.44392099 7.50 85.32936606 83.34573105 8.35 85.32936606 86.32118357 Overlay of % Solubility for Unheated/ Heated Casein % Solubility 100 90 80 70 60 50 40 30 20 10 0 0.00 2.00 4.00 6.00 8.00 10.00 ph % Solubility of Unheated Casein % Solubility of Heated Casein Figure 4: Overlay % solubility of unheated/heated whey at various ph s.
V. CALCULATIONS: Final unheated whey concentration at ph 2.5: Line of best fit à A= 1.0635x + 0.1117 x= (A-b)/m x=(0.392 0.1117) / 1.0635 = 0.264 mg/ml % unheated solubility of whey at ph 2.5: 0.264 mg/ml / 0.75 mg/ml * 100 = 35.14% Final unheated casein concentration at ph 2.7: Line of best fit à A= 1.2099x + 0.0438 x= (A-b)/m x= (0.462 0.0438) / 1.2099 = 0.3456 mg/ml % unheated solubility of casein at ph 2.7: 0.3456 mg/ml / 0.50 mg/ml * 100 = 69.13% VI. DISCUSSION: For lab 6, we created a standard curve using a Bradford Standard Assay. The purpose of standard curves is to ultimately find the concentration of an unknown protein. The word standard generally means the use of something we already know similar to a standard solution. In this case, our standard was bovine serum albumin (BSA). In table 1, one can see how the [BSA] was diluted with various amounts of water. After the standard curve was plotted on Microsoft Excel, we directed the program to include a linear line of best fit. For my group s standard curve (whey), one can see that the line of best-fit equation was 1.0635x + 0.1117. To determine final protein concentration for whey, one would solve for x with a given absorbance. Then, to determine the % solubility of whey, one would take the final protein concentration value and divide that by the original protein concentration (0.75 mg/ml for why). Finally, just multiply this value by 100 to arrive at a percent value. This process is illustrated in the calculations portion of the lab report.
For this report, there were not many deviations for our standard curve. When observing table 1, one can see that the standard deviations are small values such as 0.01414 for tubes 2 and 3. The reason why there appears to be little deviations is because our sample size was 2. In figure 4 (casein), we expect that the unheated/heated curves would be more or less on top of each other. This is due to the fact that casein is fairly stable in the presence of heat. Our data seems to support this prediction. In figure 3 (whey) we expected the heated curve to have a slightly lower solubility than the unheated one. Our data does not really support this prediction (except at around ph 4). For the most part the heated curve is slightly above the unheated curve. Some sources of error could include a mediocre ph meter or cross-contamination when recording values. Also, it is interesting to note that the % solubility of whey is fairly stable from ph s 2-8. The % solubility stays between the ranges of 30%-40%. This makes sense because whey is heat unstable and ph stable. The isoelectric point is where the protein carries no net electrical charge. At this point, we would expect solubility to be the lowest. Water prefers to react with charged/polar molecules. At around ph 4, we can see in figure 4 that casein s % solubility is almost at 0%. This observation makes sense because casein is insoluble at that ph. In figure 3, we can see that whey s lowest % solubility is also around that point. However, it is important to note that it is still a lot more soluble than casein in comparison. For % solubility, we would expect whey to have more of a response to heat change. This is because whey proteins can be changed and destroyed at high temps. For most of our data points, the data did not follow this prediction. At ph 8.60, the % solubility for heated whey was 63.73% while the unheated was at 55.20%. At ph 6.50, the heated whey had 42.16% solubility while the unheated was at 29.88%. This was not expected, sources of possible error was listed above. Conversely, we would expect casein to have more a response to ph change. It is insoluble at around ph 4.76. This point can be seen in figure 4, but the ph is around 4 instead of 4.76. From ph 2.70, to 3.65, the solubility decreased drastically. The % solubility
difference between unheated and heated was 69.13%-->6.81% and 66.82%--> 4.66%, respectively. VII. CONCLUSION: In this lab we used the Bradford Assay standard curve in order to determine the protein concentrations of whey and casein. Then we used those values to determine the % solubility of those proteins. In general, we should expect that casein s % solubility is more affected by ph change while whey s % solubility is more affect by heat change. Our data suggested this theory for casein but not so much for whey. This experiment was important because food scientists want to know which proteins are more soluble in certain environmental stresses such as heat/ph. This ultimately helps them improve aspects like storage quality and taste. VIII. QUESTIONS: 1) At ph 4.50 whey was the least soluble, and at ph 3.65 casein was the least soluble. Caseins normally exist as micelles while whey proteins are more globular. At this point the casein s glycomacropeptide (GMP) is neutralized by the ph change. Whey are a little more stable at this ph because have higher levels of cysteine which result in more disulfide bonding. These bonds stabilize the protein. 2) For the whey, the heated solutions actually had a higher solubility, slightly. However, this is the opposite of what should really happen since whey is heat sensitive. Sources of error could include mislabeling, dirty ph meter, etc. For casein, we didn t observe any noticeable differences. Casein is not heat sensitive. Secondary structure is dictated by the primary sequence of amino acids while tertiary/quaternary structure are dictated by the environment. The various structures are important because the bonds between them such as H-bonds can affect solubility. Also, a certain way a protein folds can expose or hide polar/nonpolar groups for whey and casein.