Lab #3: Ion Exchange Chromatography

The protein molecules in many foods provide the amino acid building blocks required by our own cells to produce new proteins. To determine whether a sample contains protein, a reagent called Biuret solution is used. Biuret solution contains copper ions. However, the chemical state of the copper ions in Biuret solution causes them to form a chemical complex with the peptide bonds between amino acids (when present), changing the color of the solution. Biuret solution is normally blue, but changes to pink when short peptides are present and to violet when long polypeptides are present.

This Lab Report is an analysis of the results of a two-part experiment. In the first part, we used a gel filtration column to separate the components of a mixture composed of protein and non-protein molecules. By doing so we hoped to obtain fractions that contained single components of the mixture, while also gaining insight into the relative molecular weight of each component compared to each other. We would then plot these fractions onto nitrocellulose paper in order to determine which fractions had protein. In the second part, we would use the fractions which we had determined had protein to conduct an SDS-PAGE. By doing so we hoped to determine an estimate on the molecular weight of the proteins present in each fraction by comparing it to a tracker dye composed of a variety of molecules of differing molecular weight.

The shape and magnitude of the UV spectra depends on the composition of amino acid in each protein. Due to the aromatic amino acid residues in the protein, the observed UV absorbance was mainly in the 240 nm to 340 nm region. In Figure 1 to 3, the maximal absorbance of each protein was approximately at 280 nm. The difference in magnitudes of the peak observed was linked to the differences in the amino acid contents in each of the proteins. The peak of lysozyme was greater than those of BSA and gelatin, because lysozyme has a greater number of tryptophan residues. Lysozyme has six tryptophan residues, whereas BSA and gelatin has two and zero, respectively (Department of Chemistry, 2014). Lysozyme has three times more tryptophan residues

This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal

For the peptide Ala-Arg-Lys-Ala-Asn-Ser-Ala-Ser, what would be the expected charges at pH 1, 7, and 13?

To improve the results from the experiment buffer solutions that were not whole pHs could have been used e.g. pH 4.5, 5.5 etc. This would have provided more reliable results as a wider range of results would have been produced. Using pHs with decimals would also help to more accurately determine the optimum pH as the optimum may have been above or below the pH stated in the hypothesis; 8. In this experiment however the optimum is taken at 8 because the graph does not rise again.

To make the buffer solution you need 0.2 mol dm-3 of Na2HPO4 and 0.1mol dm-3 of citric acid this will give 100cm3 of buffer. Here is how to get the different pH in the buffer solutions:

Describe the significance of using 9 mM sodium chloride inside the cell and 6 mM potassium chloride outside the cell, instead of other concentration ratios. The reason for the use of 9mM of sodium chloride is because 3 ions are ejected for every 2 K that are added. K hangs outside the cell unless actively transport inside the cell.

The results recorded in (table 30) and (figure 32) Indicated that Cu+2 activated the enzyme at 0.01M concentration by 1.2 fold and the activity gradually decrease by increasing the metal concentration to 0.1M with activity 51.29U/mg protein. Na+ ion activate the enzyme when added with 0.1M by

In macromolecular mass spectrometry, electro spray ionization (ESI) is used to generate multiple charged ions of a protein or protein complex, especially producing ion signals with a roughly Gaussian-shaped and charge-state distribution. If the mass resolution is enough, there are adjacent peaks in the spectra which could be observed presented the charge and the mass of the analyte being determined. Viruses and capsids are subjected to ESI mass spectrometry (11, 12) Nevertheless, due to insufficient mass resolution, precise masses are hard to be determined. Because of highly improved technology, it is well prepared to analyze in vitro-assembled HBV capsids characterized in a wide range of particles tests. It analyzed two capsids of cp149 variants: the subject and control group, 3C→A ( cysteine-free )and 61C(cysteine included),respectively. Then to test the masses of molecular under a spectrum of high mass resolution and separated charge states, both caspids reveals the similar pattern. These spectra produced masses of both capsids respectively, and the measurements are quite closed to the expected masses of capsids, consisting of 90 and 120 dimers, responding to the subject and control group. No other particle with which fewer or more than 90 and 120 dimers was

For the ion-exchange, there are two different methods, cation and anion; both depend on the pH mobile phase, the stationary phase, and the eluent used. For cation exchange, the mobile phase typically has an acidic pH, around pH 3, and the stationary phase of the column uses a material with an overall negative net charge. The eluent used to separate the whey from the column is then a liquid with a pH that is neutral or slightly

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

The pH of a solution is the measure of the concentration of charged Hydrogen ions in that given solution. A solution with a pH lower than seven is considered to be acidic. A solution with a higher pH is a base. It is very important for organisms to maintain a stable pH. Biological molecules such as proteins function only at a certain pH level and any changes in pH can result in them not functioning properly. To maintain these constant pH levels, buffer solutions are used. A buffer solution can resist change to small additions of acids or base’s. A good buffer will have components that act like a base, and components that act like an acid.

For this experiment, titrations on a weak acid, acetic acid, and a buffer were performed. Acetic acid was titrated with NaOH in order to observe the half-equivalence point as well as the equivalence point. Then, the buffer and the buffered acetic acid solution prepared faced additional titration with NaOH and HCl to evaluate the differing buffering effects following the addition of a strong acid and strong base. Finally, the buffer’s buffering capacity was calculated. If the experiment were to be repeated, it would be interesting to observe the buffering effects following a titration between a weak base and a buffer instead with greater concentrations. The change in the concentration following the preparation of buffer with a weak base and its conjugate acid would pose for an interesting experiment to observe an increase in the buffering capacity.

The titration curve of the unknown exhibited many characteristics, such as equivalence points, pKa of ionizable groups, isoelectric point, and buffer regions, that are particularly distinct to lysine. For unclear reasons, the pH during the titration did not reach the pH for pure 0.2 M NaOH nor 0.2 M HCl and normal equivalence points expected at two extreme ends of the titration curves for all amino acids were not observed. The titration of a phosphate buffer showed that the buffer capacity is directly proportional to the molarity of the buffer. However, our results showed that although the initial pH of the phosphate buffer was less than the pKa value, the measured buffer capacity was higher towards acid than base. The accuracy of the pH meter and calibration process was questioned under assumptions that the pH of the prepared phosphate buffer was actually above pKa.