Isolation and Characteristics of Proteins by Hydrolysis of Intact Proteins

Diane Michelle A. Battung, Ralph Alvino T. Cabasag, Emille Jean S. Caragay, Kristine A. Castro, Gwen Steffi B. Co Group 2 2F-Pharmacy General Biochemistry Laboratory

ABSTRACT
A protein is a naturally occurring, unbranched polymer in which the monomer units are amino acids. More specifically a protein is a peptide in which at least 50 amino acids residues are present. Proteins can be classified into two types: fibrous and globular. Fibrous proteins are proteins in which peptide chains are arranged in long strands or sheets. Globular proteins are proteins that tend to fold back on themselves into compact spheroidal shaped units. Globular proteins do not form intermolecular interactions between protein units and are more easily solubilized in water as colloidal suspensions than fibrous proteins are. In this experiment, all the samples are in globular form. The experiment is to isolate casein from skimmed milk. Having a sample of 20.0gmilk, following the procedures, the group obtained 6.5g of casein. Computing weight of casein obtained over weight of the milk multiply by 100%, the group achieved a weight percentage of 32.5%.

INTRODUCTION
Casein is a phosphoprotein, which has phosphate groups are attached to some of the amino acid sidechains. These are attached mainly to the hydroxyl groups of the serine and threonine moieties. Actually, casein is a mixture of at least three similar proteins, which differ primarily in molecular weight and amount of phosphorus they contain (number of phosphate groups). Casein exists in milk as the calcium salt, calcium caseinate. This salt has a complex structure. It is composed of , , and caseins which form a micelle, or a solubilized unit. Neither the nor the casein is soluble in milk, singly or in combination. If casein is added to either one, or to a combination of the two, however, the result is a casein complex that is soluble owing to the formation of the micelle. A structure proposed for the casein micelle is shown on the following page. The casein is thought to stabilize the micelle. Since both and casein are phosphoproteins, they are precipitated by calcium ions. The casein in milk can also be clotted by the action of an enzyme called rennin. Rennin is found in the fourth stomach of young calves. However, both the nature of the clot and the mechanism of clotting differ when rennin is used. The clot formed using rennin, paracaseinate, contains calcium. calcium

Enzymatic, Acidic and Alkaline Hydrolysis of intact protein was also done in this experiment. The 20 amino acids commonly found as hydrolysis products of proteins contain an -carbonyl, an -amino group, and a distinctive R group substituted on the -carbon atom. Hydrolysis of the protein and analysis of the product are done to obtaini nformation about their compositions. Hydrolysis can be carried out by treating the protein with acid, alkali or proteolytic enzymes. In acidic hydrolysis, hydrochloric acid will be used. It vaporizes when heated then comes in contact with the protein sample and hydrolyzes the sample. On the other hand, sodium hydroxide will be used on the intact protein for alkaline hydrolysis. Enzymatic hydrolysis is the test to the presence of proteases. It is also known aspeptidases or proteolytic enzyme. These occur naturally in all organisms. They cut peptide bonds of proteins at specific amino acid residues producing peptide fragments and some free amino acids. Currently, there are six classes of peptidases specific to either N- or C-side of the followingamino acid residues: serine, threonine, cysteine, aspartate, glutamate, metallopeptidases.

EXPERIMENTAL
A) Materials and Compound Used

reaction and separation and identification of amino acids by thin layer chromatography.

RESULTS AND DISCUSSIONS

y y y y y y y y y Isolated casein 6M HCl 4M NaOH 1M HCl 1M NaOH Saturated protease solution Red and blue litmus paper Hard glass test tubes 250-mL beaker An 8N H2SO4 was added to the isolated casein and was autoclaved for five hours at 5 psi. Before autoclaving, the casein was colour beige and it was in its solid form and liquid 8N H2SO4 was added in the Erlenmeyer flask. After autoclaving, the solution was already a dark brown liquid. The hydrozylate was diluted with 15 mL of distilled water and was subjected toneutralization by adding solid Ba(OH)2 until reaching ph 5. While saturated Ba(OH)2 was added until it reached ph 7 after the solution reached ph 5. Its initial pH was 0.87because the solution contained 8N H2SO4 upon autoclaving. Acid hydrolysis is the most common method for hydrolyzing a protein sample before amino acid analysis. The acid hydrolysis technique can contribute to the variation of the analysis due to complete or partial destruction of several amino acids. Tryptophan is destroyed; serine and threonine are partially destroyed; methionine might undergo oxidation; and cysteine is typically recovered as cystine (but cystine recovery is usually poor because of partial destruction or reduction to cysteine). Acid hydrolysis of proteins before analysis disturbs the original equilibrium between the two compounds so that the composition of the hydrolysate no longer reflects that of the protein

B) Procedure 1. Acid hydrolysis of intact protein Add 5mL 6M Hydrochloric acid to 0.5gisolate protein in a hard glass test tube then label. Stopper the tube with cotton and submit to the instructor for autoclaving (15 psi for 5 hours).Take note the appearance of the mixture after autoclaving. Add 10mL distilled water to the mixture then transfer to a 250-mL beaker. Neutralize the mixture with 1M sodium hydroxide for characterization tests and thin layer chromatography. 2. Alkaline hydrolysis of intact protein Add 10 mL 4M sodium hydroxide to 0.5gisolated protein in a hard glass test tube then label. Stopper the tube with cotton and submit to the instructor for autoclaving (15 psi for 5 hours).Take note the appearance of the mixture after autoclaving. Add 10mL distilled water to the mixture then transfer to a 250-mL beaker. Neutralize the mixture with 1M hydrochloric acid for characterization tests and thin layer chromatography. 3. Enzymatic hydrolysis of intact protein Place 1g/100mL distilled water protein mixture. Mix 10mL of protein mixture and 10mLof saturated protease solution. Alternatively,0.050g of protease may be added directly to 50mLprotein mixture. Add 10mL 0.1M phosphate buffer pH 7.5. Incubate the tube in water bath maintained at 35-40C (depending on the source of enzyme)for 60 minutes. Alternatively, digestion may be carried out overnight at room temperature. Allow the mixture to cool before using it for tests for qualitative color

REFERENCES
From books Crisostomo, A.C., et al. 2010. Laboratory Manual in General Biochemistry . Quezon City, Philippines: C&E Publishing Inc. pp.20-22 From internet [1] http://courses.chem.psu.edu/chem36/Web%20S yn06/Exp112Syn06.pdf [2] http://homepages.ius.edu/dspurloc/c122/casein.htm