Isolation of Casein and Albumin

ISOLATION OF PROTEINS: CASEIN AND ALBUMIN Michael Jon P. Alvarez, Francesca Eirylle M. Arriola, Sieren B. Baguingan, Pauline Anne E. Basco, Danielle Therese M. Bermudez and Leah Therese A. Camangon Group 1 2B Medical Technology Biochemistry Laboratory

ABSTRACT Selected proteins were isolated from their commercially available sources using different isolation techniques for each. The group was assigned to isolate the proteins casein and albumin from a sample of skimmed or non-fat milk by means of isoelectric precipitation and heat denaturation, respectively. The group used a 50 mL sample of Nestle nonfat milk which was first heated to 40 oC, after which 10% acetic acid was added dropwise until a curd-like precipitate was formed, indicating that the isoelectric pH of casein has been reached. The precipitated casein was filtered by gravity filtration and appeared as a white amorphous precipitate. The filtrate from which albumin was isolated appeared as a cloudy yellowish liquid. It was placed in a 75 oC water bath. No precipitate was formed as the liquid stayed in the water bath longer than the indicated 5 minutes.

INTRODUCTION Milk is one of the substances that are abundant in different types proteins. The primary protein component of milk is a protein group called caseins. Casein is the general term for a family of related phosphoproteins. These make up about 80% of the proteins in cow’s milk and around 2045% of the proteins in human milk. The casein family of proteins consist of several types of casein, namely -s1, -s2, ß, and 6 [3]. These form a multi-molecular granular structure called a micelle, along with water and some salts [1]. They bear a specific amino acid composition that is essential to for growth and development, which makes milk one of the most essential components of a healthy human diet. One of the significant properties of caseins is that it precipitates or coagulates at pH 4.6. Aside from this, certain albumins can also be found in milk in the form of lactalbumins. Albumins are globular proteins that are soluble in water and in dilute salt solutions and have the ability to become denatured and coagulated by heat application [2]. A specific example of a lactalbumin is -lactalbumin, which is classified as a whey protein. -Lactalbumin plays an important role in the synthesis of lactose and is essential in the process of milk synthesis [1]. Once casein has been isolated from the milk and the milk has been made acidic, lactalbumin can be precipitated out by heating the mixture. In this experiment, several proteins were isolated from their commercially available sources which is needed in order to be able to study their structure, properties, and different functions of proteins. There are various methods by which proteins can be isolated that involve different principles. One of these methods is isoelectric precipitation. The isoelectric pH (pI) is the pH value at which the protein has zero net charge. Isoelectric precipitation is done by adjusting the pH of the protein source until the pI is reached. This reduces the solubility of the protein, since it

is unable to interact with the other components of its source, which will then lead to its precipitation [1]. Another method by which proteins can be isolated is by heat denaturation. Denaturation of proteins involves the disruption of its secondary and tertiary structure. Denaturation reactions, however, are not strong enough to break the peptide bonds that constitute to the protein’s primary structure, so the amino acid sequence remains intact. Denaturation by heat application can be used to disrupt hydrogen bonds and nonpolar hydrophobic interactions since heat increases kinetic energy which causes intense, rapid vibration of molecules, leading to the breakage of the said bonds [4]. Other protein isolation techniques or principles are salt-induced precipitation wherein high concentration of salt causes the solubility of the protein to decrease greatly (a process called salting-out) [6], and isolation by simple difference in solubility in substances such as water. The first part of this experiment aims to isolate casein and albumin from skimmed milk by isoelectric precipitation and heat denaturation respectively, gluten from wheat flour by difference in solubility and myoglobin from beef heart by salt-induced precipitation.

EXPERIMENTAL A. Sample used Nestle Non-Fat Milk

B. Procedure 1. Isolation of casein from skimmed milk 50 mL of the liquid milk sample was placed in a 100-mL beaker, and then heated on a hot plate while maintaining the temperature at 40oC. Upon reaching this temperature, 10% acetic acid was added dropwise into the milk sample while the solution was stirred gently after every 5 drops. This was done continuously until the pH of the

solution reached 4.6, or when a precipitate or curd was observed. The precipitated casein was filtered out by gravity filtration and the filtrate was set aside for the isolation of albumin.

2. Isolation of albumin from skimmed milk The filtrate which was previously obtained from the filtration of casein was heated in a 75 oC water bath for 5 minutes. After this, the liquid in the solution was decantated off to obtain the precipitated albumin.

The filtrate that the group obtained from filtering out casein appeared as a slightly cloudy yellowish liquid. After immersing it in a heated water bath, the protein albumin was supposed to be precipitated out of the solution. However, the group failed to arrive at this result and it was suspected to be due to the fact that the mixture was overheated as it was left in the water bath for over 5 minutes. The group proceeded to filter the heated solution, retrieving only the same yellowish liquid that was initially separated from casein and heated.

RESULTS AND DISCUSSION The group was able to obtain the following results after following the procedure for isolating casein and albumin from the milk sample:

2. after water

Table 1. Isolation of assigned proteins PROTEIN Casein Albumin

DESCRIPTION Clumps of yellowish white solid precipitate (amorphous) Yellow cloudy liquid

Upon addition of acetic acid to the milk sample, the group was able to observe formation of curd. As previously discussed, isolation of proteins via isoelectric precipitation allows the protein to precipitate out of its source or medium by lessening its solubility once it reaches its pI. This indicates that at the point wherein curd started to form, a sufficient amount of acetic acid has been added to adjust the pH of the sample to around 4.6 which is the pI of casein. After filtering the solution, the collected casein precipitate was observed to be a white amorphous precipitate.

of the

Figure Albumin filtrated heating in bath The principle behind the separation albumin involves

denaturation or the breaking of bonds of the protein upon application of heat. When the protein solution that came from the casein filtrate was heated, the albumin was denatured and had lost its secondary and tertiary structures. The general process of denaturation can be explained in the following diagram: Figure 3. Effect of denaturation on the structure of a protein As a result of the alteration of its structure and

consequently some of its properties, the protein loses its solubility in the solution, making it possible for it to be isolated. Figure 1. Filtered casein precipitate

REFERENCES [1] Hurley, Walter L. Milk Composition: Proteins. http://ansci.illinois.edu/static/ansc438/Milkco mpsynth/milkcomp_protein.html 03/28/2016 [2] Minard, R. Isolation of Casein, Lactose and Albumin from Milk. http://courses.chem.psu.edu/chem36/New %20Syn%2036%20pdf/Exp112.pdf 03/29/2016 [3] n.a. Milk Protein. http://www.milkfacts.info/Milk %20Composition/Protein.htm 03/29/2016 [4] Ophardt, Charles E. Denaturation of Proteins. http://chemistry.elmhurst.edu/vchembook/56 8denaturation.html 03/28/2016 [5] Young, Derek R. Isoelectric Precipitation. http://www.rpi.edu/dept/chem-eng/BiotechEnviron/PRECIP/precpph.html 03/28/2016 [6] Young, Derek R. Salt Induced Precipitation of Proteins. http://www.rpi.edu/dept/chemeng/Biotech-Environ/PRECIP/precpsalt.html 03/29/2016