EXPERIMENT 1: ISOLATION AND PURIFICATION OF PROTEINS

Bea Lorraine Acosta and Karl Desquitado

Locker No. 12

Chemistry Department, Xavier University - Ateneo de Cagayan, Cagayan de Oro

City
Date Performed: June 21, 2016
Date Submitted: June 30, 2016

ABSTRACT

Proteins are compounds that are very important and most diverse.
Proteins slightly vary from one another due to their different structures,
sequences, and configuration. Milk and beans were the samples used in which
the proteins were to be isolated from. Casein from milk was obtained by
isoelectric precipitation in which the pI of the protein must be obtained in order
to be precipitated. Protein from bean was obtained through denaturation. The
percent proteins (w/w) of the samples were not obtained due to the incomplete
purification of the proteins.

INTRODUCTION

Proteins are organic compounds that are very important to the cells (1).
Proteins are considered the most diverse group of important biological
substances (2). Proteins are polypeptides composed of linear chains of amino
acids (3). Each protein has different structures, sequences, and configuration
which result to a slight variation on the physical and chemical properties of
different proteins. Since proteins tend to exist all at once in a single sample or
source, purification of proteins is not a simple process.
In this experiment, the proteins were isolated through isoelectric
precipitation. In isoelectric precipitation, the pH of the solution was increased or
decreased in order to precipitate the protein. The pH value at which the protein
precipitates is known as the isoelectric point (pI). The value of the pI is the pH
where the protein is generally least soluble and eventually precipitates (4).
In this experiment the proteins from milk and beans are isolated and
purified.

MATERIALS AND METHODS

Isoelectric Precipitation of Casein from Milk

Twenty mL of skimmed milk was placed in a 250mL beaker and then
constantly mixed with 0.1M hydrochloric acid until the pH of the solution was 4.6.
To avoid extreme acidity in any portion of the solution, the acid was added at a
rate of 5mL per minute of mixing. A suspension was produced. The suspension
was allowed to stand until the precipitated casein settles down. The supernatant
liquid was discarded. The precipitate was washed with water until the washings
were no longer acidic to litmus. The precipitate was then dried between filter
papers.

Isolation of Bean Protein

 One cup of beans were sorted, washed and then soaked in water for 24
hours. The beans were ground finely with water and then blended with more
water using a blender. The blended beans with water were now called milk. The
milk was expressed through cheesecloth, the insoluble residue was discarded.
The protein was (extracted) precipitated by adding 1M of acetic acid until
complete precipitation. The mixture was allowed to stand for some time and then
strained through the cheesecloth. The water was squeezed out through the cloth
and then the excess water was removed by pressing the precipitate between
filter papers.

RESULTS AND DISCUSSION

Several proteins can be found in milk but most of these proteins have very
low concentrations. Casein consists 80% of the milk and is present as calcium
salt and calcium caseinate. The pI of casein was approximately 4.6 and at this pH
value, casein had zero net charge (5) and was precipitated. Since milk had a pH
between 6.5 and 6.7, the casein micelles will have a negative net charge but are
stable. Upon addition of the acid to milk, on the outer surface of the micelle the
negative charges were neutralized and then the neutral protein was precipitated
(4).

Figure 1. Casein from milk.

The protein in beans was extracted by adding acetic acid. Upon the
addition of the acetic acid the protein was immediately denatured, along with
precipitation and thus immediately stopping the activity of other present
enzymes (6).
 Figure 2. Bean protein.

CONCLUSION

In a compound several different proteins are present. Isolating and

purifying all proteins is not a simple process. Casein and bean protein were
successfully extracted. The percent protein (w/w) of the samples were not
obtained due to incomplete purification of the proteins.

LITERATURE CITED

1) Bailey, R. (2016). What Are Proteins?. About.com Education. Retrieved 28 June

2016, from http://biology.about.com/od/molecularbiology/ss/proteins.htm

2) The Chemistry of Biology: Proteins. (2016). Infoplease.com. Retrieved 28 June

2016, from http://www.infoplease.com/cig/biology/proteins.html

3) Tropp, B. (2012). Molecular biology. Sudbury, Mass.: Jones & Bartlett Learning.

4) Isoelectric Precipitation of Proteins: Casein from Milk (Theory) : Biochemistry

Virtual Lab I : Biotechnology and Biomedical Engineering : Amrita Vishwa
Vidyapeetham Virtual Lab. (2016).Vlab.amrita.edu. Retrieved 28 June 2016,
from http://vlab.amrita.edu/?sub=3&brch=63&sim=158&cnt=1
5) Isoelectric Precipitation. (2016). Eng.umd.edu. Retrieved 28 June 2016, from
http://www.eng.umd.edu/~nsw/ench485/lab6c.htm

6) Martnez-Maqueda, D., Hernndez-Ledesma, B., Amigo, L., Miralles, B., &

Gmez-Ruiz, J. (2012). Extraction/Fractionation Techniques for Proteins and
Peptides and Protein Digestion. Proteomics In Foods, 21-50.
http://dx.doi.org/10.1007/978-1-4614-5626-1_2