test tube, all the information was written at the top so that way the spectrophotometer wouldn t read the sharpie.

Transcription

1 Mary McKenna 11/7/2013 A07 Thursday 9am SL, LR Lab 6 Protein Solubility and Spectrophotometry I. Purpose/Objective The purpose of this experiment was to obtain solubility information from whey and casein proteins using spectrophotometry and observe how the difference absorption values corresponded to their different protein structure. II. Introduction When studying proteins, their functional properties are taken into consideration. Functional properties are the chemical and physical properties that determine the usefulness and behavior of proteins in food systems during processing, storage and consumption. These functional properties are related to the protein structure, for instance if the structure changes the properties will change as well. Some specific property examples are curd formation by casein in milk, the stickiness dough like consistency of wheat gluten, and the foaming that is caused by beating an egg white. Since those proteins act in a specific way, the food industry could potentially manipulate proteins in order to create more consistent products. For this experiment specifically, solubility of bovine serum albumin was looked into as well as whey and casein proteins. Solubility is an important criterion determining how the food will interact with water. It depends on the composition and ratio of polar to nonpolar amino acids, their sequence, and concentration. In this experiment, the bovine serum was used to create a protein concentration standard curve by adding varying concentrations of water and Bradford reagents. The next solubility test that was done was with casein and whey protein, and the percent solubility of each protein was then determined. It is important to the food industry to know how soluble each protein is because whey and casein proteins are currently being added to nutritious drinks. The way these proteins breakdown and act within the body is all due to their specific structure, and if the food industry wanted to make proteins that worked the same way, they could modify different proteins to mimic whey and casein. III. Procedure The procedure followed for the experiment is found in Principles of Food Composition Laboratory Manual (2013) Experiment 2, Acidity in Foods, pages Modifications included making a 400 ml beaker a waste container for the Bradford Reagent. Also, for making the standard concentration curve, 0.8 mg/ml of BSA in a 1.5 ml conical tube was used. Finally, when labeling the

2 test tube, all the information was written at the top so that way the spectrophotometer wouldn t read the sharpie. IV. Data/Result Table 1. Concentration and absorbance data for standard curve made with BSA Table 2. Absorbance data reflecting solubility of heated/non-heated whey and casein protein in different ph environments, also calculated % solubility of the two proteins.

3 Figure 1. Figure 1 depicts the standard curve of the Bradford Assay, includes the equation of best fit. Figure 2. Figure 2 shows the % solubility of heated and non-heated whey protein as a function of ph.

4 Figure 3 Figure 3 shows the % solubility of heated and non-heated casein protein as a function of ph. V. Calculations 1. Final casein 3.42 ph Abs y = 1.978x = 1.978x = 1.978x x = x= g/100ml 2. % Solubility casein = g/100ml x 100 = 23% 0.05 g/100ml 3. Final Whey 3.31 ph Abs y = 1.978x = 1.978x = 1.978x x = 0.19 x = g/100ml 4. Solubility whey = g/100ml x 100 = 19% 0.1 g/100ml

5 VI. Discussion By looking at the data from the experiment, different characteristics where seen between the two types of proteins, and also the differences between the heated and non-heated versions. For the whey protein, both the heat and non-heat samples were more soluble at around ph However, the heated sample for whey had the most solubility at around 35% solubility, where the peak for the non-heated was about 30% solubility. On the other hand, casein had a more drastic variance. The heated sample of the casein showed had the most percent solubility at a more acidic ph of 5, and the non-heated sample peaked at a slightly basic ph of around 7.5. A thing to note though is that with the casein, both the heated and the non-heated had around 40% solubility when it reached its maximum. By comparing whey and casein, it can be observed that casein will reach around the same high peak of percent solubility for heated and non-headed samples but it is all dependent on the ph. Whereas the whey protein, the peak solubility happens around the same ph for the heated and non-heated, however the heated is a lot more soluble. The relationship between solubility and the isoelectric point is that a protein is least soluble at this point. There is no net charge to be interacted with so therefore it is not as soluble. The standard curve that was used (figure 1) provided the equation in order to determine the percent solubility of the whey and casein proteins. There might be some source of error if the solutions were not mixed properly, if that happened, the equation and therefore the percent solubility would be not as precise. VII. Conclusion The protein solubility experiment showed that ph and heat could change certain characteristics of different types of proteins. Since the functional properties changed, the protein that once reacted the exact same now reacts differently. The spectrophotometry used was very helpful since it allowed the percent of solubility to be calculated from its absorption reading. The overall purpose provided a clear picture on how food chemists can change properties of proteins. VIII. Questions 1. The least soluble ph for whey was a heated sample at 7.5 ph, and for casein it was a non-heated sample of 5.4 ph. At the isoelectric point, there is no charge on the protein so it wouldn t want to react with water, it would prefer to react with other proteins. Since it will not react with water, it isn t soluble. 2. Minimum solubility happens around the same time in the heated and non-heated whey samples. They are both soluble at around 5.5 ph. For casein, the heated is more soluble at a ph around 5, and the unheated around 7.5. For both proteins however, the minimum solubility occurs around a ph of Molecular structures influence the degree of solubility because different confirmations can affect the

6 extent of the interaction of water on the available polar sites. These conformational changes are due to a change of ph and heat, which both were done in this experiment. The ph affects the net charge on proteins thus affecting their ionic relation with water.