ISOLATION OF CASEIN FROM SKIMMED MILK AND SEPERATION AND IDENTIFICATION OF AMINO ACIDS BY PAPER CHROMATOGRAPHY

B, E., *C, K., C, K., C, S., D, A., 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

Abstract Proteins are insoluble at their isoelectric pH, the pH at which the net charge is equal to zero. This can be done by subjecting the proteins to extremes of heat and pH, and different denaturing solvents. Casein was isolated from low fat milk through isoelectric precipitation by altering its pH to 4.6 using 10% acetic acid at 40oC. Then albumin was isolated by heat denaturation. Paper chromatography was performed for the separation and identification of amino acid standards based on the polarities. The non polar (hydrophobic) amino acids were closer to the solvent front. The polar uncharged amino acids were in the middle and the polar charged amino acids in the lowest part. The result shows that the non polar (hydrophobic) amino acids have the highest affinity with the mobile phase while the polar charged amino acid has a low affinity with the mobile phase.

Introduction Proteins are the most abundant organic molecules found in living cells. They are polymers of amino acids. There are 20 different amino acids commonly found in proteins. Proteins are insoluble at their isoelectric pH, the pH at which the net charge is equal to zero. This can be done by subjecting the proteins to extremes of heat and pH, and different denaturing solvents. Denaturation alters the proteins function, demonstrating a relationship between structure and functions. Proteins may be classified broadly in two general categories: fibrous and globular. Globular proteins are those that tend to fold back on themselves into compact units that approach nearly spheroidal shapes. These types of proteins do not form

intermolecular interactions between protein units (Hbonds and so on) as fibrous proteins do, and they are more easily solubilized as colloidal suspensions. There are three kinds of proteins in milk: caseins, lactalbumins, and lactoglobulins. All are globular. Casein is a phosphoprotein, which has phosphate groups are attached to some of the amino acid side chains. 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. Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; therefore casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated) and the neutral protein precipitates: Ca2+Caseinate + 2HCl Casein + CaCl2[1] Isoelectric casein and some other forms of casein is insoluble in water but may be converted to water soluble caseinates by dispersion in water and adjusting the pH to 6.7 with alkali, usually NaOH to yield sodium caseinate.[2] Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat. The second most abundant protein types in milk are the lactalbumins. Once the caseins have been removed, and the solution has been made acidic, the lactalbumins can be isolated by heating the mixture to precipitate them. The typical albumin has amolecular weight of about 41,000. A third type of protein in milk is the lactoglobulins. They are present in smaller amounts than the albumins and generally denature and precipitate under the same conditions as the albumins. The lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own immune system has developed.

One of the objectives of the experiment is to perform hydrolysis in preparation for the qualitative color reactions. The 20 amino acids commonly found as hydrolysis products of proteins contain an -carbon group, and a distinctive R group substituted on the carbon atom. Hydrolysis of the protein and analysis of the products are done to obtain information about about their compositions. Hydrolysis can be carried out by treating protein with acid, alkali, or proteolytic enzymes. Chromatography comes from the Greek word chroma, means colour and graphein, to write. Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. There are different kinds of chromatography namely: paper chromatography, thin layer chromatography, gas chromatography, liquid chromatography, high performance liquid chromatography, affinity chromatography, colum chromatography. In paper chromatography, the stationary phase is very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents. [3] For the identification and separation of amino acids, paper chromatography can be used. It is a method of separating the components of a mixture based on their differential affinity for two chemicals, which are the stationary phase and the mobile phase. Polarity of proteins can be determined through this method. Materials and Apparatus Isolation of Casein from skimmed milk: 1. Non fat milk

2. 3. 4. 5. 6.

10% acetic acid Thermometer Ph meter/ indicator Funnel and filter paper/ cheese cloth Hot plate

using a cheese cloth. The decantate was set aside for the isolation of albumin and the residue was dried. The weight % of the casein was calculated

Isolation of Albumin from skimmed milk: 1. Decantate from the isolation of casein 2. Thermometer 3. Hot plate 4. Water bath Hydrolysis of intact protein: 1. Isolated Casein 2. 6M HCL 3. 4M NaOH 4. 1M HCL 5. 1M NaOH 6. Saturated protease solution 7. Red and blue litmus paper 8. Hard glass test tube 9. 250 ml beaker Seperation and identification of amino acids by paper chromatography: 1. Amino acid standards: 2% w/v tryptophan, arginine, proline, cysteine, serine, aspartic acid, tyrosine, histidine, glycine, and alanine 2. Acid, basic, and enzymatic hydrolysates 3. 1-Butanol: acetic acid: water (4:1:5) 4. 1% ninhydrin solution in spray bottle 5. Filter Paper 6. Chromatography chamber (1-L beaker covered with a watch glass) 7. Capillary tubes Methodology 1. Isolation of Casein from skimmed milk The 75 ml Non fat milk was heated up to 40 o C, 10% acetic acid was added until the solution reaches 4.6 pH or until casein has precipitated. The solution was then filtered

Figure 1. Non fat milk at its isoelectric pH of 4.6

Figure 2. Filtered Casein from Non fat milk 2. Isolation of Albumin from skimmed milk The decantate was heated in the water bath for 5 minutes at 75 o C. then the liquid was decant off from the precipitated albumin.

Figure 3. Filtered Albumin 3. Acid Hydrolysis of Intact Protein To the 0.5g of isolated protein in a hard glass, 5 ml 6 M HCL was added and the tube was labeled. The tube was plugged with cotton and was autoclave at 15 psi for 5 hours. After autoclaving, the mixture was added 10 ml distilled water and was transferred into a 250 ml beaker. Lastly, the mixture was neutralized with NaOH. 4. Basic Hydrolysis of Intact Protein To the 0.5g of isolated protein in a hard glass, 5 ml 6 M NaOH was added and the tube was labeled. The tube was plugged with cotton and was autoclave at 15 psi for 5 hours. After autoclaving, the mixture was added 10 ml distilled water and was transferred into a 250 ml beaker. Lastly, the mixture was neutralized with HCL. Figure 4. autoclaving Basic Hydrolysate after

5. Enzymatic Hydrolysis of Intact Protein 1 g/100 ml distilled water protein mixture was prepared. Then, 10 ml of protein mixture and 10 ml of saturated protease solution was mixed and was added 10 ml 0.1 M phosphate buffer at pH 7.5. Lastly, the tube was incubated in a water bath at 35-40 o C for 60 minutes. 6. Seperation and identification of amino acids by paper chromatography The origin was drawn as a pencl line across the filter paper with a 1.5 cm margin from the bottom of the longer edge of the plate. 13 equidistant points was marked on the line for spotting of the amino acid standards and 3 hydrolysate samples. The standard was applied 5 times and the samples 10 times using capillary tubes. Then the plate was placed inside the preequilibrated chamber and was covered and allowed for the solvent to ascend undisturbed. The plate was removed when the solvent front is approximately 0.5 cm from top edge of the plate and the solvent front was marked with a pencil. The chromatogram was air dried and was sprayed with 1% ninhydrin reagent. It was then placed inside an oven for 1-3 mins.

Figure 3. Basic Hydrolysate before autoclaving

Then the spots where marked and the RF value was calculated.

Percentage Error

12.84%

Cows milk contains approximately 3.5% protein, 80% casein (2.8%) and 15% whey protein.[4] The theoretical weight of Casein from 75 mL of Milk is 2.18 g and the experimental weight was 1.9 g which is equal to 2.43% percentage yield. Percentage yield Figure 5. Paper Chromatography of amno acids and Hydrolysate of Casein Results and Discussion Isolation of Proteins Casein is the protein that was isolated from non-fat milk through adding acetic acid. When acetic acid was added to the non-fat milk mixture at a controlled pH level, yellowish white precipitate was produced. That process is called isoelectric precipitation. Precipitation occurred because the protein has already reached its isoelectric point wherein its net charge equaled to zero. Once the caseins have been removed, and the solution has been made acidic, the lactalbumins can be isolated by heating the mixture to precipitate them. Table 1 Data obtained from the isolation of Casein Casein Volume of Milk Weight of Casein Percentage Yield White Crumbs 75 mL 1.9 g 2.43% Yellow % casein = g Casein x 100 g Sample = 1.9 g x 100 75ml x (density = 1.04) =78g =2.43% Percentage error = Theoretical yield actual yield X 100 Theoretical yield = 2.18g 1.9g X100 2.18g =12.84% Hydrolysis of Intact Protein Hydrolysis of intact proteins breaks covalent bonds of amino acids in the presence of water and structures that are lost in hydrolysis are the secondary, tertiary, and quaternary structures. Hydrolysis can be classified into three types such as acid, alkaline, and enzymatic hydrolysis. In acid hydrolysis, there is complete hydrolysis of peptide bonds and no racemization. However, one disadvantage is the destruction of tryptophan to humin (blackprecipitate). In alkaline or basic hydrolysis, there is also complete hydrolysis of peptide bonds but the disadvantage is arginine would form ornithine and urea. In enzymatic hydrolysis, bonds are broken through peptidases or proteolytic enzymes. These are enzymes that

break long chain-like molecules or proteins into shorter fragments and eventually into their components, the amino acids. Common proteolytic enzymes are pepsin (which breaks all peptide bonds), trypsin (which breaks arginine and lysine), chymotrypsin (which breaks phenylalanine, tyrosine and tryptophan),amino peptidase (which breaks amino terminals),and carboxypeptidase (which breaks carboxyterminals). Advantages of performing the enzymatic hydrolysis are: it is fast and specific and it does not destroy amino acids. On the other hand, one disadvantage is that it only requires certain temperatures.

proline not a true amino acid shows up as yellow serine tryptophan tyrosine

0.43

0.27 0.66 0.45

Table 3 Experimental Rf Value Amino acid alanine arginine aspartic acid cysteine glycine Rf value 0.30 0.21 0.34 0.29 0.30 0.20 0.46 0.33 0.59 0.47 0.63 0.57 0.20

Paper Chromatography

Figure 5. Paper Chromatography of amno acids and Hydrolysate of Casein Table 2 Standard Rf value of amino acids Amino acid alanine arginine aspartic acid cysteine glycine histidine Rf value 0.38 0.20 0.24 0.4 0.26 0.11

histidine proline serine tryptophan tyrosine Acid Hydrolysate Base Hydrolysate Enzyme Hydrolysate

The results of the paper chromatography are based on the polarity of amino acids. Tryptophan, a hydrophobic non-polar amino acid, moved farthest from the base line. On the other hand, histidine, a polar amino acid, moved least from the base line. The mobile phase in the solvent system is the butanol and the acetic acid. Tryptophan having the farthest traveled amino acid means that it has a high affinity to acetic acid and butanol. Histidine has a high affinity to the stationary phase, which is water. Looking at the chromatogram, most of theamino acids that can be seen far from the baseline belong to the 1st group of amino acid, the hydrophobic non-polar amino acids. On the other hand, the amino acids seen near the baseline belongs to the 2nd and 3rd group of amino acid which are the polar uncharged and polar charged amino acids respectively. Butanol, a polar protic solvent with a relative polarity of 0.552, carries the non-polar amino acids up the chromatogram because even though it is polar like water, it is less polar than it for water has a relative polarity of 1. Acetic acid, also a polar protic solvent with a relative polarity of 0.648, carries the polar amino acids up the chromatogram. Due to their quantitativedifference in the solvent system mixture which is 4:1 or four (4) measures of Butanol for everyone (1) measure of acetic acid, non-polar amino acids are favored than polar amino acids. The amino acids reacted with the 1% Ninhydrin solution giving them distinct blue and violet colors except proline which gives a yellow color. Cysteine though, had a slight yellow coloration because it was probably contaminated by proline during the spotting of amino acid standards. For the acid and the basic hydrolysate, both of them can be found near the base line of the chromatogram which means they have a high affinity to the stationary phase when treatedwith ninhydrin solution, they gave a bluish brown color.

The acid hdrolysate and the basic hydrolysate have high Rf value while, the enzymatic value has a low Rf value. Conclusion Casein was isolated from low fat milk through isoelectric precipitation by altering its pH to 4.6 using 10% acetic acid at 40 degrees Celcius. The amount of Casein obtained was a little different to the theoretical amount could be because of the possible contamination of the equipment used, or the inaccuracy of the platform balance. Then albumin was isolated by heat denaturation. Paper chromatography was performed for the separation and identification of amino acid standards based on the polarities. The non polar (hydrophobic) amino acids were closer to the solvent front. The polar uncharged amino acids were in the middle and the polar charged amino acids in the lowest part. The result shows that the non polar (hydrophobic) amino acids have the highest affinity with the mobile phase while the polar charged amino acid has a low affinity with the mobile phase. The RF value of the standard to the experimental is a little different because of the possible contamination of equipment; the solvent was disturbed, or the inaccurate application of the sample. References [1] Pavia, L., Saunder, K. ,& Saunder, E. (1990). Introduction to Organic Laboratory Techniques: A Microscale Approach. Retrieved from http://courses.chem.psu.edu/chem36/Web %20Syn06/Exp112Syn06.pdf

[2] McSweeney, P., & Fox, P. (2013). Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects, (4th Edition). Retrieved from http://books.google.com.ph/books?id=2Vd DAAAAQBAJ&pg=PA51&lpg=PA51&dq=mell ander+study+about+casein&source=bl&ots =z1U-IV78os&sig=x7tku6cp4SMuu5OOHQ2LXMWON8&hl=en &sa=X&ei=TQ2vUqK5OcK5iQefjoGIAg&ved= 0CEEQ6AEwBA#v=onepage&q=mellander% 20study%20about%20casein&f=false [3]Shokair, S. (2000) Chromatography Retrieved from College of Veterinary Medicine & Animal Resources Department of Clinical Studies Toxicology & Forensic Medicine. Website: http://www.google.com.ph/url?sa=t&rct=j &q=&esrc=s&source=web&cd=6&cad=rja& ved=0CEIQFjAF&url=http%3A%2F%2Fwww. kfu.edu.sa%2Fen%2FSpaces%2Fsalshakair% 2FDocuments%2Fchromatography.ppt&ei=ySvUvHWHWWiQeUpIAY&usg=AFQjCNH1xsqsqtn4b9Jy 4R-KyUngtabIFA&bvm=bv.57967247,d.dGI [4]Beckman coulter (2013) retrieved from https://www.beckmancoulter.com/wsrport al/bibliography?docname=IB-18070.pdf