Sari Bornstein November 4, 2010 Thursday PM E.Y. Lab #4: Protein Functionality- Solubility and Foam Formation PURPOSE/OBJECTIVE: The purpose of this lab was to compare the two main classes of milk proteins, casein and whey, based on one of their function properties, solubility, in relation to different phs and temperature. INTRODUCTION: In this lab, we performed a Bradford Assay Standard Curve. This tells us a known protein concentration in relation to its absorbance. Using this known (BSA), we can find the concentration of an unknown protein solution based on the absorbencies observed. In this case, the protein solutions were casein and whey, two of the major components in milk protein. Based on the results of absorbency of the unknown protein solution, we can determine the percent solubility of each protein at a specific temperature and/or ph by using the line of best fit of the Bradford Assay curve. The Bradford Assay curve can determine what our concentrations are of our unknown and then the percent solubility. As stated in the lab manual, most proteins produce a typical ph- solubility curve with minimum solubility in the isoelectric region, where positive and negative charges are equal and net charge is zero (p. 36). At this ph point, the protein is least reactive and therefore, insoluble. The objective of this lab was to see how solubility changes in relation to ph changing and temperature changing. This lab showed us a way to study proteins and when their environment alters, how their functionality alters as well. This is important in the food industry to make certain foods, such as whey, precipitate at a certain temperature based on its isoelectric point. For example, in the making of certain cheeses, whey is precipitated out of cheese. This is what gives some cheeses a certain taste and texture. 1
PROCEDURE: The procedure followed for the experiment is found in FST101A- Food Composition Laboratory Manual (Fall 2010) pages 35-47. No modifications were performed. DATA/GRAPHS: Table 1: Concentration and absorbance data for standard curve made with BSA Tube # [protein] (mg/ml) Absorbance 1 0.0 0.0 2 0.1 0.43 3 0.2 0.44 4 0.4 0.562 5 0.8 0.96 Table 2: Absorbance data reflecting solubility of heated and non- heated solutions of whey protein Absorbance ph Heated Non- heated 2.66 0.400 0.460 3.64 0.478 0.472 4.51 0.333 0.442 5.54 0.492 0.494 6.51 0.486 0.484 7.53 0.548 0.520 8.62 0.508 0.514 Table 3: Calculated data showing % solubility of whey protein for heated and non- heated, as a function of ph ph Absorbance (y) protein concentration (g/100ml) (x) % solubility (%) heat No heat heat No heat heat No heat 2.66 0.400 0.460 0.0311 0.0356 31.1 35.6 3.64 0.478 0.472 0.0371 0.0367 37.1 36.7 4.51 0.330 0.442 0.0256 0.0344 25.6 34.4 5.54 0.492 0.494 0.0382 0.0384 38.2 38.4 6.51 0.486 0.484 0.0378 0.0376 37.8 37.6 7.53 0.548 0.520 0.0459 0.0404 45.9 40.4 8.62 0.508 0.514 0.0395 0.0399 39.5 39.9 2
Table 4: Absorbance data reflecting solubility of heated and non- heated solutions of casein Absorbance ph Heated Non- heated 2.48 0.684 0.490 3.52 0.436 0.347 4.49 0.212 0.202 5.48 0.644 0.400 6.53 0.662 0.548 7.52 0.394 0.484 8.51 0.550 0.566 Table 5: Calculated data showing % solubility of casein protein for heated and non- heated, as a function of ph ph Absorbance (y) protein concentration (g/100ml) (x) % solubility (%) heat No heat heat No heat heat No heat 2.48 0.684 0.490 0.0532 0.0381 106.3 76.2 3.52 0.436 0.347 0.0339 0.0270 67.8 53.9 4.49 0.212 0.202 0.0165 0.0157 33.0 31.4 5.48 0.644 0.400 0.0501 0.0311 100.1 62.2 6.53 0.662 0.548 0.0515 0.0426 102.9 85.2 7.52 0.394 0.484 0.0306 0.0376 61.2 75.2 8.51 0.550 0.566 0.0427 0.0440 85.5 88.0 3
Bradford Assay Standard Curve Absorbance @ 595 nm 1.2 1 0.8 0.6 0.4 0.2 0 y = 1.2867x R² = 0.91377 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 concentration of BSA (mg/ml) Figure 1: The Bradford Assay standard curve plotting absorbance (at 595 nm) versus concentration (in mg/ml) of BSA, including the line of best fit % solubility 50 45 40 35 30 25 20 15 10 5 0 Solubility of Heated and Non- heated Whey 0 2 4 6 8 10 ph Figure 2: % solubility of heated and non- heated of whey protein as a function of ph Heat Non- heat 4
120 100 Solubility of Heated and Non- heated Casein % solubility 80 60 40 20 Heat non- heat 0 0 2 4 6 8 10 ph Figure 3: % solubility of heated and non- heated of casein protein as a function of ph CALCULATIONS: Final whey concentration y= 1.2867x 0.400 = 1.2867 (x) x= 0.3109 mg/ml 1g 0.3109mg 100mL =0.0311 g/100ml 1000mg % solubility of whey 0.0311g /100mL 0.1g /100mL 100% = 31.1% Final casein concentration y= 1.2867x 0.684 = 1.2867 (x) x= 0.5316 mg/ml 1g 0.5316mg 100mL = 0.0532 g/100ml 1000mg % solubility of casein 0.0532g /100mL 0.05g /100mL 100% = 106.3% 5
RESULTS/DISCUSSION: In this lab, a Bradford Assay Curve of BSA was used to determine an unknown protein concentration, casein or whey. Based on the concentration of protein at different ph and temperatures, the percent solubility can be determined. First, the absorbencies of different concentrations of BSA were plotted in a Bradford Assay curve (concentration versus absorbance), and the line- of- best- fit was used to find the concentration of either whey or casein, based on their absorbances at different phs at room temperature or heated. In my Bradford Assay curve, somehow the same volume of BSA was put in vials 2 and 3, so these two vials ended up having around the same absorbances. When plotted, this produced a line with a low R 2 value. Eliminating vial 2 produced a better line of fit and a larger R 2 value. A reason R 2 wasn t 0.999 was because of error. There may have been error is measuring the liquids with the micropipette, or in mixing each vial, or even with the amount of wait time required before measuring each absorbance. Nevertheless, a standard curve was produced to help determine the concentrations of whey and casein in relation to differences in temperature and ph. Then when the concentrations are known, percent solubility can be calculated. When percent solubility is graphed versus ph, it produces a parabolic- like curve, where the lowest point on that curve is the isoelectric point. The isoelectric point is the ph at which the molecule has a net charge of zero, meaning it is least reactive at this point and therefore has minimum solubility. This can be seen in both figure 2 and figure 3. Because heat was added to whey, at a ph of 4.6 is where whey is least soluble, meaning the protein has started to uncoil, bringing its inner nonpolar amino acids towards the outside. Therefore, the protein will have less charge and unable to be soluble in solution (low % solubility). Because casein is ph sensitive, at a ph of 4.6, the molecule will have an overall zero charge, so it will not be attracted to other charged molecules. Therefore casein can be precipitated due to its low % solubility. CONCLUSION: In this lab, we determined the solubility of two different proteins when changing their association with heat and different ph. A Bradford Assay was used to determine the ph at which casein and whey were least soluble. By using a protein of known concentration, this method seemed very effective. It was interesting to see the variable environments of a 6
protein in relation to how they reacted in solution. This is very important to the food industry in determining what proteins to put into food due to ph or temperature favorability that gives them the best taste or texture. In addition, I know the properties of proteins at different ph and temperatures are important in determining the taste and texture of cheese. One improvement for later labs would to be the use of glass test tubes for the heat- treated protein solution so that the temperature can be raised and we could analyze the properties at both proteins at higher temperatures. Using plastic test tubes limited us from doing that. QUESTIONS: 1. The point when casein and whey are least soluble is at their isoelectric point, which can easily be seen in figure 2 and figure 3. The isoelectric point for whey is calculated to be at a ph of 4.6. The isoelectric point for casein is calculated to be at a ph of about 4.6 as well. It is at the isoelectric point that the protein is not very reactive because its net charge is zero. Therefore, the molecule does not have any charges to attract or mix well with other molecules, hence minimum solubility. This is why whey protein is soluble at ph 4.6 and casein can be precipitated at ph 4.6. 2. As seen in figure 2, the heated whey has an isoelectric point around ph 4.6, but the non- heated whey was close to linear. This proves that as temperature changes, the solubility of whey changes. Therefore, whey is heat sensitive. A contributing factor to this is whey s structure. Whey is a globular protein that situates non- polar amino acids towards the center of the globular protein and the polar amino acids around the outer layer to come into contact with other polar molecules, including water. However when heated, the protein can start to denature, which causes a conformational change in the structure. This can expose the non- polar amino acids and break disulfide bonds, hence making it insoluble. As seen in figure 3, heated and non- heated casein solutions were both affected, therefore we know it is ph sensitive. Casein s structure is opposite of that of whey. Casein is a micelle that is highly structured, which is why (at least in this lab), casein was undisturbed by heat. On the outside of the micelle, GMP tails interact with surrounding environment while the non- polar amino acids face towards the center. With the altering of ph, the GMP tails can be 7
removed, exposing a hydrophobic molecule that will be insoluble in water. At a ph of 4.6, this is what s occurring. 8