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Abstract:

The process of protein purification should be done from the very first step. This will enable understanding of the protein structure and function. Appropriate techniques should be chosen to achieve the process of purification. We shall discuss four different techniques in this report. The first technique is fractionation. In this process, salt is used for salting out and acetone is used to reduce the dielectric constant of water, which will result in elution of the amylase protein. The tube containing the fraction of the dialyzed was tested for starch. Iodine was added and the solution remains colorless indicating absence of starch. To determine the presence of glucose, 3, 5-dinitrosaliylic acid was reduced to 3-amino, 5-nitrosalicylic acid. The value 570nm was needed to calibrate a curve for the test of alpha-amylase activity. In order to achieve this, the enzyme used was amylase and substrate was starch. A Michaels -Menten curve was drawn followed by a Lineweaver Burk plot. The Km value was found to be 1.19 and velocity 76.923 µmole/ min. Another method of purification is ion exchange chromatography. In this method, a mixture of resin is used as a medium by which molecules are passed at a set of conditions. The resin that was used for the project is DEAE sepharose the positive charge while amylase is negative. After testing the result showed, that2 tubes had a transparent color and the rest blue. The results show that amylase was hydrolyzed and blue indicates the absence of amylase in the tube. In Polyacrylamide Gel Electrophoresis, which is another method of purification, sodium dodecyl sulfate was used to denature proteins by making their charges negative. Separation occurs based on molecular weight. The amylase in the gel had three bands instead of one and this because it might be not purified. However, the unknown protein had only one fragment, which means that it is pure and its size was about 43.65 kDa based on the marker on the scale

Introduction

The aim of the experiments conducted was to find differences in the various methods used to purify proteins. Proteins are nutrients required by the body. They are macromolecules that help in building and repairing worn out tissues. They are however not found in their purified form and thus studying them requires purification. Knowledge about the structure and function has led to the development of various methods of purification. Using the knowledge of side chain, they can be separated according to their hydrophilicity or hydrophobicity features. This method is known as fractionization. Separation of proteins according to size is known as Gel permeation. Separation according to the charges is known as exchange chromatography.

Fractionalization method depends on precipitation by the use of a salt such as ammonium sulfate and sodium sulfate or an organic solvent. The hydrophilic end of the amino acid occupies the outer surface while the hydrophobic end occupies the inner area. Upon addition of salt, there is a competition between the salts and proteins for water thus decreasing the solubility of proteins. The proteins are then precipitated. Another name for this process is salting out.

To study more about protein functions, an enzyme assay was done. The amylase obtained from fractionization is required to hydrolyze starch. Iodine was added to give a color change and to show when the reaction was complete. In the presence of starch, iodine turned blue. As the reaction proceeds, a colorless color will be observed until all the amylase is hydrolyzed.

A second experiment was done to determine the glucose concentration using three, 5- dinitrosalicylic acid (DNSA) method. This method shows the presence of a free anomeric carbonyl group (C=O) which is found in all simple sugars. This technique also includes oxidation of aldehydes or keto groups. By the end of experiment 3-amino, 5-nitrosalicyclic acid will be formed as a result of the reduction of 3, 5- dinitrosalicylic acid (DNSA). The product has a maximum absorption of 540nm at which a reaction will be observed. The next step in the procedure is to find out reducing sugars by alpha-amylase and to determine its activity. α -Amylase stimulate the hydrolysis of (1-4) linkages of starch resulting in two sugars maltose and isomaltose. This reaction is also done using 3, 5-dinitrosalicylic acid (DNSA) and the absorption will be at 540 nm.

In ion chromatography, proteins are separated depending on the charge. The matrix contains electrostatic interactions that have an opposite charge to the proteins. This method has two options, anion exchange chromatography where the positive resin( example DEAE) react with negatively charged proteins and cation exchange chromatography where the negative resin (example sepharose) react with positively charged ions. In this experiment, it will be a cation exchange chromatography to separate cytochrome c and hemoglobin

All proteins have a pI value with a neutral charge. Any slight change in the pH value as compared to the pI value results in a difference in charge. For example a above the pI makes the protein to acquire a negative charge while below is it become positive. With this knowledge, the protein is isolated using a salt gradient that has a higher affinity to the matrix. This results in the protein being eluted. At a high pH, the aim of using a salt is to prevent the protein to be denaturized.

Finally, the Polyacrylamide gel electrophoresis method is used to separate large molecules. Electrophoresis is the process of separation of molecules by charges. When a current is applied, the molecules move to the opposite pole from their charge. A gel is used to transmit the current. Example of currents used is agarose gel which is used for large molecules (this gel have large porous so make the migration easy for these large and complex molecules), the other gel is Polyacrylamide used for small oligonucleotides and proteins (this gel is easy for the migration of these small molecules because it has a small porous). Buffering the pH of the solution makes it easier to maintain the mobility of proteins. In special cases, however, migration may be due to molecular weight and not charge Dodecyl sulfate is a detergent that denatures proteins by making their charges negative and the migration will be based on their molecular weights. That is why the experiment is named as SDS-PAGE. These are a few methods used to isolate and purify proteins.

Procedure:

A- Fractionation of amylase protein

a- Ground 5g of germinating seeds, then add 20ml of 0.1 M sodium phosphate buffer of PH7. Then filtered the homogenous solution.

b- Precipitation by ammonium sulfate:

· Put 05.ml of saturated Ammonium sulfate and 1 ml of protein in respective microfuge tubes. Mixed the components of the tubes by use of vortex and incubated them for 10 minutes under ice.

· For 10 minutes i centrifuges at 1400 rpm of tubes for almost 10 minutes.

· I extracted the supernatant

· Repeated the same procedure for subsequent experiments

· Allowed the pellet to dry.

· Dissolved half of 0.1 sodium phosphate

· Transferred the mix into an 8cm dialysis bag and left it at a temperature of 4⁰C for 24hours.

· Solution was transferred from the dialysis bag and stored.

c- Precipitation by acetone:

· The same procedure as preparation of Ammonium Sulfate but now with 0.5 Cold Acetone being used as an alternative of sodium phosphate buffer.

B- Amylase assay using iodine test for starch

· Made use of two test tubes

· Add 0.5ml of 5% concentered starch solution in a tube then add 0.5ml of 0.1 M sodium Phosphate buffer.

· Added Iodine in the first tube

· Added 5 drops of protein in second tube then incubated the solution for 5 minutes. Added iodine after incubation.

C- Determination of glucose by DNSA:

· 2% of glucose solution was used in preparation of 0.03, 0.05, 0.07, 0.1, 0.2 and 0.3% diluted solutions.

· Solutions of 0.5 ml of DNSA and 0.5 ml of 2M NaOH were added to all the tubes.

· Mixed the solutions with vortex {the tubes were subjected to direct heat for 5 minutes having been covered by glass beads}

· After the removal of glass beads the tubes were cooled at room temperature.

· Added 0.5 ml of 40% sodium potassium tartrate and mixed the tubes well.

· Absorbance was done at 540 nm

· Recorded the readings.

D- Determination of Reducing Sugars Produced from Hydrolysis of Starch by Alpha-Amylase and Amylase Enzyme Activity

· Dissolved 10 mg of alpha-Amylase in a 10 ml of 0.1 M sodium phosphate buffer pH 7.0.

· The procedure used for determination of glucose by DNSA was repeated.

E- Ion exchange chromatography:

Buffers required are:

1. 0.05M Sodium phosphate pH7.0

2. 0.1 M NaCl was added to 0.05M Sodium phosphate pH7.0

· Opened the column and 1 ml fractions collected.

· Loaded the column

· Added 1 ml of the protein to the column

· The sample has to be passed through the column.

· Added 10 ml of 0.05M sodium phosphate pH7.0 buffer

· Continued collecting the fractions.

· After the addition of all the buffers, 10 ml 0.05M Sodium phosphate pH7.0 with 0.1M NaCl

· Ensured that fractions collections progressed till entire buffer was eluted.

· After tuning off of the column, alpha-amylase was tested by use of iodine.

F- SDS-PAGE:

· The technician prepared gel and placed the gel in a container.

· Buffer solution was filled up in the reservoir.

· To make wells visible, comb was placed in the gel

· Loaded 5 µl of the ladder with the good number 1

· 15 µl of the mixture of dye and sample was placed in the second well

· 15 µl of the tested unknown was loaded in the 3rd well.

· Results and Discussion:

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· Qualitative amylase assay using iodine test for starch:

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Control

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Supernatant

Fraction of dialyzed protein

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· Figure 1: amylase assay with iodine on different amylase precipitation concentration.

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· In the test of amylase presence, three Eppendorf tubes were used. The first tube had just 0.5 ml of 5% starch, 0.5 ml of 0.1 M sodium phosphate and two drops of iodine and depicted blue color upon completion of incubation process. The second tube had a 50 µl of dialyzed protein, 0.5 ml of 5% starch, 0.5 ml of 0.1 M sodium phosphate and two drops of iodine. Upon completion of the incubation process the Eppendorf color disappeared indicating the presence of amylase in the solution. This is as a result of amylase hydrolyzing the starch into monosaccharide hence disappearance of the color. For the third tube, after centrifuging the precipitated protein from fractions. A sample of supernatant was taken to test presence of amylase. Results indicated negative presence of amylase since the solution maintained its color.

· Part 1: Determination of Glucose concentration by 3,4-dinitrosalicylic acid (DNSA) method:

· The experiment was done using different dilutions of glucose solutions. A graph was plotted for absorbance against different concentration of glucose that was diluted. The graph indicates the concentration increases the Absorbance value increase. The concepts behind this experiment are that when the 3,5-dinitrosaliylic acid reacts with glucose, it can be reduced to 3-amino, 5-nitrosalicylic acid by the help of amylase enzyme under basic condition, which will absorb maximally at 570 nm. The value that was obtained from the calibration curve of glucose solution can test the alpha-Amylase activity.

· part 2: Determination of Reducing Sugars Produced from Hydrolysis of Starch by Alpha-Amylase and Determination of Amylase Enzyme Activity

The concentration of starch 0.6 was measured two times, the first value was measured by using the alpha-amylase that extracted two ago from the seed, and the other value was prepared by the TA. The activity that prepared by the TA had lower velocity value possible error is the preparation of sample by the TA was prepared a long time ago, this may have denatured the of the sample. It cannot be used to compare or measure the accuracy of the experiment that performed.

The SDS-PAGE gel had three wells each was loaded with different protein. Lane 1 had Alpha amylase that was extracted from seed couple of weeks before the experiment. The amylase showed three fragments that indicate that the alpha-amylase was contaminated with another component and was not fully pure because if the sample were pure it would show only one fragment. That happened because the sample was obtained directly from fractions that prepared a couple weeks ago and without purifying with other protein technique such as ion exchange chromatography. Lane 2 has the unknown protein and only one fragment was shown which indicates the protein is pure. The molecular mass can be calculated by using lane 3, which is the marker.

RF= Distance moved by the fragment of protein /Distance travel by the dye

The unknown molecular mass was determined by using the standard curve of the marker protein. The molecular weight of the unknown is 43.65 kDa. Using the standard as references and using the RF, the unknown molecular mass was calculated and the value indicates that molecular mass if lighter which allow the migration of the band. Because if the band was heavy or greater in molecular Wight the band will not travel as far, cause the heavier band the less migration occurs. The Polyacrylamide gel has small pore and allow the small size travel faster and farther from the loading place while the bigger size migrate slowly and not far

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· . Conclusion:

The aim of the report was to discuss and analyze the various purification techniques. These are placed in line with the properties of proteins. After isolation and purification using fractionation method, which was based on hydrophobic and hydrophilic ends, the sample was available for study for their properties. The ion exchange does not fully purify the protein from other contaminants. In the SDS-PAGE experiment, the alpha-amylase was in three stages. This is a clear indication of the purity of the sample. After isolation and purification, the sample should be used within two weeks to avoid denaturing and loss of value thus wrong results in the next experiment.