Lee 1 Yuen Wai Lee 11/5/2012 Tuesday afternoon lab M.S.P., EB.A.

Lab 4- Protein functionality protein solubility and foam formation I. PURPOSE/ OBJECTIVE The purpose of this lab is to evaluate the functional properties of protein in milk like its solubility and concentration by using a standard curve.

II.

INTRODUCTION Proteins functional properties are the physical and chemical properties that affect their behavior and usefulness during production and consumption. Generally, protein can be used in binding, hydration, sensory, surface, matrix formation and structural purposes. One of the properties of protein we were analyzing in this lab is the solubility of protein. It is important as it can be used to determine the interaction of water and other functional properties also depend on the solubility of protein such as foaming, emulsification, and gelation. The solubility of protein on the conformation, surface change, composition , ratio of polar to non- polar amino acids, and the solution such as its ion type, concentration, pH, and temperature. For protein to be soluble in water it has to be able to form hydrogen bonds between protein and water which means it should be polar to water. Hydrogen bonding with water can connect the polar groups in different regions of proteins. Polar amino acids with ionized side chains bind the largest amount of water while non- ionized ones bind in intermediate amount and hydrophobic amino acids bind little or no water. In other words, the solubility generally increases with the increasing polarity if protein surface and decreasing molecular weight. Moreover, by changing the protein conformation, the availability of polar sites can be affected also. The compact globular conformation to a random coil can result in increasing solubility due to exposure of hidden peptide bonds and amino acid side chains that can interact with water whereas exposure of hydrophobic groups can cause a decrease in solubility. These conformational changes can be caused by change in temperature, pH or

Lee 2 other environmental change. Proteins usually produce a pH-solubility curve with minimum solubility in the isoelectric region where the positive and negative charges are the same. The water binding and solubility when net charge is zero is minimized. Casein protein and whey protein under investigation in this lab is found in milk. Caseins are phosphoproteins that precipitate at pH 4.6 and whey proteins are soluble at pH 4.6. Casein proteins come in micelle form, which are colloidal aggregates, while whey protein exist in solution and are globular. Caseins have distinct cluster of hydrophobic and negatively charged region along the peptide chain and a few cystine residues which exist in open, extended form.

III.

PROCEDURE The procedure for this experiment is found in Principles of Food Composition, Laboratory Manual, FS&T 101A (2012) Experiment 4, Protein functionality protein solubility and foam formation, Pages 43-46. Modifications include using 0.25N, 0.125N, and 0.625 N NaOH and HCl to adjust the pH of protein. For the standard curve, 12.5 L BSA and 87.5L water was added instead of 0.010 mL of BSA and 0.090mL of water into tube2; 25.0 L BSA and 75.0L water was added instead of 0.020 mL of BSA and 0.080mL of water into tube3; 50.0 L BSA and 50.0L water was added instead of 0.040 mL of BSA and 0.060mL of water into tube4; 100.0 L BSA and 0L water was added instead of 0.080 mL of BSA and 0.020mL of water into tube5 and wait for 5 minutes. The cloudy protein samples were centrifuged for 15 minutes at maximum RPM instead of 20 minutes.

IV.

DATA/ RESULT Table 1: Concentration and absorbance data for standard curve made with BSA

Tube Conc. BSA (mg/ml) Absorbance 1 0 0 2 0.1 0.143 3 0.2 0.308 4 0.4 0.552 5 0.8 1.005

Lee 3 Table 2: Absorbance of heated and non- heated casein and whey proteins in designated pH

pH 2.58 3.65 4.45 5.6 6.45 7.65 8.54

Whey Protein absorbance Heated non- heated 0.648 0.506 0.634 0.406 0.596 0.486 0.418 0.5 0.74 0.608 0.86 0.702 0.732 0.718

Table 3: Absorbance of heated and non- heated casein proteins in designated pH

pH 2.7 3.64 4.45 5.61 6.44 7.5 8.66

Casein Protein absorbance Heated non- heated 0.203 0.214 0.326 0.366 0.117 0.131 0.561 0.52 0.564 0.554 X X 0.508 0.494

Table 4: % solubility of the whey proteins, heated or non- heated, as a function of pH

pH 2.58 3.65 4.45 5.6 6.45 7.65

Whey Protein % solubility Heated non- heated 50.0425 39.08 48.9613 31.35 46.0267 37.53 32.2805 38.61 57.1473 46.95 66.4144 54.21

Lee 4 Table 5: % solubility of the casein proteins, heated or non- heated, as a function of pH

pH 2.7 3.64 4.45 5.61 6.44 7.5 8.54

Graph 1: Standard curve

Casein Protein % solubility Heated non-heated 31.35 33.05 50.35 56.53 18.07 20.23 86.65 80.32 87.11 85.57 x x 78.46 76.30

Standard Curve
1.2 1 Absorption 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 Conc. BSA (mg/ml) Abs
Best fit line ) (Abs (Abs)

y = 1.2949x R = 0.9925

Lee 5 Graph 2: % solubility of heated and non- heated whey protein

Whey Percent Solubility vs. pH

70.0000 60.0000 Percent Solubility (%) 50.0000 40.0000 30.0000 20.0000 10.0000 0.0000 0 2 4 pH 6 8 10 Heated % solubility non-heated % solubility

Graph 3: % solubility of heated and non- heated casein protein

Casein Percent Solubility vs. pH

92.0000 82.0000 72.0000 Percent Solubility (%) 62.0000 52.0000 42.0000 32.0000 22.0000 12.0000 2.0000 -8.0000 0 2 4 pH 6 8 Heated % solubility non-heated % solubility

Lee 6 V. CALCULATIONS Final heated whey concentration at pH 2.58: Y= 1.2949x, where y is the absorbance at pH 2.58, according to table 2, its 0.648 0.648= 1.2949x X= 0.5004 mg/mL The final heated whey concentration is 0.50mg/mL.

% Solubility heated whey at pH2.58: % Solubility= (Final whey conc. (mg/mL)/ original protein conc. (mg/mL))*100% = (0.5004mg/mL/0.1g/100mL*1000mg/1g) = 50.04% The % Solubility heated whey at pH2.58 is 50.04%.

Final heated casein concentration at pH 2.70: Y= 1.2949x, where y is the absorbance at pH 2.70, according to table 3, its 0.203 0.203=1.2949x X=0.1568 mg/mL The final heated casein concentration is 0.16mg/mL.

% Solubility heated casein at pH2.70: % Solubility= (Final casein conc. (mg/mL)/ original protein conc. (mg/mL))*100% = (0.5004mg/mol/0.05g/100mL*1000mg/1g) = 31.36% The % Solubility heated whey at pH2.70 is 31.36%.

VI.

DISCUSSION From graph 1, the standard curve was y= 1.2949x and the R2 is 0.9925. From the best fit line equation, the protein concentration can be determined by substituting the absorbance into the equation to solve for x which is the concentration. The % solubility of protein can thus be found also by dividing the protein concentration by the original protein concentration. In general, the absorbance increases when protein concentration increases. The % solubility also increases when the concentration of protein increases. The R2 value shown in graph 1 indicates the deviation of the standard curve. The larger the value, the smaller the deviation is. From graph 1, the R2 value is 0.9925. This indicates that

Lee 7 the protein concentration against absorbance was not too far away from the best fit line. The point at 0.2mg/mL for protein concentration is the farthest point from the best fit line; this error can be caused by an uneven mix sample tested. The isoelectric point for casein protein is at pH which means the protein has a lowest solubility at this point and tends to precipitate. From table 5, it was shown that the % solubility for both heated and non- heated casein at pH 4.45 is the lowest. This matches the isoelectric point of casein protein in the lab manual. Casein has a lowest solubility at this pH as it tends to precipitate when the charged GMP is removed by enzymatic action and being neutralized. When GMP is removed, the hydrophobic micelle remained would coagulates as hydrophobic interactions occur. The repulsion between casein by negative charge GMP is removed by positive ions. When the repulsion is gone, the casein protein will precipitate which in turn decreasing its solubility in solution. While most casein proteins precipitate at pH 4.6, whey protein remains as solution at this pH. According to table 4, the heated whey protein sample the lowest solubility occurs at pH 5.6 and that of non-heated one is pH 3.65. The isoelectric point for whey protein is different as it is a more diverse group than caseins. It has a more uniform distribution of hydrophilic and hydrophobic amino acids so it has a more compact and globular structure. The whey problem is more sensitive to heat and the globular structure unfolds when heated exposing the disulfide bridging inside. In general, the whey protein is more soluble in extreme pH. According to table 4, whey protein has a lowest solubility at pH3.65 when not heated and at pH5.6 when heated. According to table 5, casein protein has a lowest solubility at pH4.45 when heated and non- heated. According to table 4, it was shown that the solubility of whey is lower when not heated and often has a higher solubility at extreme pH. According to table 5, it was shown that the solubility of casein is lower when heated and at lower pH. The casein protein may have denatured at extreme pH and temperature and would precipitate when the repulsive electrostatic force is removed between casein micelles. The absorbance of casein at pH 7.5 was not provided by the other group as there was an error that occurred during the experiment. The lowest solubility of casein protein was expected to be found near pH 4.6 as it is the isoelectric point for the protein. Also, the data for absorbance for BSA for making the standard curve was not able to be recorded as error has occurred during the preparation of sample. One of our group mates accidentally used a p1000 mechanical pipette instead of a p200 one to transfer the solution which in turn making the solution too dark for finding the absorbance within the range provided.

Lee 8 VII. CONCLUSION In this lab, standard curve was used to find out the protein concentration. The method was successful in analyzing the effect of temperature and pH on protein solubility. The accuracy of the experiment would be increased if a more accurate pH meter is used. As water is a weak buffer, it is harder for the pH meter to determine the accurate pH of the solution. This may cause errors in the lab data collected.

VIII.

QUESTIONS

1. Using your data, identify the pH at which whey and casein proteins are least soluble. Describe the condition of the protein at this pH that causes this point of minimum solubility. According to table 4, the whey is least soluble at pH at pH3.65 when not heated and at pH5.6 when heated. According to table 5, casein protein is least soluble at pH4.45 when heated and nonheated. According to table 4, it was shown that the solubility of whey is lower when not heated and often has a higher solubility at extreme pH. The hydrophobic groups were hidden during this pH so the solubility of protein would decrease. Casein is least soluble at this pH more precipitates form when the charged GMP is removed by enzyme and being neutralized. When GMP is removed, the hydrophobic micelle remained would coagulates as hydrophobic interactions occur. The repulsion between casein by negative charge GMP is removed by positive ions. When the repulsion is gone, the casein protein will precipitate which in turn decreasing its solubility in solution. 2. At their respective points of minimum solubility, contrast the degree of solubility between whey and casein proteins for both heated and non- heated protein solutions. Discuss how molecular structures influence the degree of solubility for each type of protein. According to table 4, the whey is least soluble at pH at pH3.65 when not heated and at pH5.6 when heated. According to table 5, casein protein is least soluble at pH4.45 when heated and nonheated. The whey protein has a different lowest solubility pH as it is a more diverse group than casein. It has a more uniform distribution of hydrophilic and hydrophobic amino acids and is more sensitive to heat. Thus it is easier to break the disulfide bonding between whey proteins and will precipitate easily at more neutral pH. The casein protein is less sensitive to heat so it is harder to break the bond between micelles. However, when the charged GMP is removed by lower pH, the repulsive electrostatic forces between micelles are also removed and more precipitate is formed and solubility is decreased.