Amanda’s Blog

Acid/base titrations can be used to determine the buffering capability of a particular amino acid. Acid or base is added dropwise to a solution containing the amino acid of interest and to a solution containing only water. Because of the buffering ability of the amino acid, the solution with the amino acid will resist a change in pH around the pK_a values of its functional groups. The solution with water is a control to compare what the pH would be without the buffer. The behavior of each solution in response to the addition of more acid or base can be analyzed with a titration curve.

The enzyme must be within a certain temperature range to function properly. At very low temperatures, the energy of a solution is very low. The molecules in the solution have less vibrational energy and tend to have fewer collisions. Because an enzyme’s activity depends on interactions between molecules of the enzyme and the substrate, the activity of the enzyme will decrease at very low temperatures. As temperature rises, the energy of the molecules is increased, therefore they have more collisions. As enzyme and substrate interactions increase, the activity of an enzyme will also increase, but only up to certain point. If the temperature continues to rise, the protein will denature, resulting in a decrease in enzyme activity. This is because the hydrogen bonds of the water external to the folded protein become disrupted. At lower temperatures, the enthalpy of the hydrogen bonds overcomes the entropy of the proteins. When the temperature rises to a certain point, these hydrogen bonds break and their enthalpic contribution is no longer great enough to overcome the entropy of the protein. This causes the protein to unfold into a more disordered form that requires less free energy.

Enzymes are proteins that accelerate metabolic reactions by binding to substrates and catalyzing their transformation into product. Enzymes are very selective and will usually only react with a single substrate by binding at its active site. When the enzyme binds to the substrate, it modifies the substrate’s geometric and electronic configuration so that less free energy is required for the substrate to reach its transition state between substrate and product. Because they are not permanently altered by the reaction, a single molecule of enzyme can catalyze many individual reactions.  Enzymes can greatly accelerate reaction rates, but their activity is limited to certain temperature and pH ranges.  Outside of these ranges, the proteins will become denatured or lose functionality.

 Taq DNA polymerase is a heat stable DNA polymerase found in the organism Thermus aquaticus. It is used in PCR because it can withstand high temperatures and remains viable after the DNA is heated to 95°C during the denaturation step. It also allows the elongation to run at a higher temperature, which increases the efficiency of replication. A drawback to using Taq is that it has a high error rate due to its lack of 3’-5’ exonuclease proofreading activity.

The polymerase chain reaction(PCR) is used to amplify specific DNA or RNA sequences through an enzymatic process. It requires upstream and downstream primers, DNA polymerase, four dNTP’s (dATP, dCTP, dGTP, and dTTP) a magnesium ion and a DNA template to be amplified. PCR is performed in three steps. In the first step, the DNA is heated to 95°C where it becomes denatured. In the second step, the DNA is cooled to 55°C for annealing of the primer. The two primers line up complimentary to the 3’ starting point of the sequence. These primers must be long enough to bind to a unique site on the template DNA. Once the DNA has lined up and annealed with the primer, the DNA is reheated in the final step to 72°C. This allows the DNA polymerase to elongate and amplify the target sequence. This cycle is repeated, producing two new fragments of product duplex DNA per template DNA.

Native gel electrophoresis does not separate effectively by molecular weight because the native gel electrophoresis technique does not use involve denaturation of the proteins.  The proteins retain their folded structure and migrate through the gel according to their mass:charge ratio. The charge of a protein is based on the ionizable side groups of its amino acids. This means that two different proteins with similar molecular weights may travel different distances due to different charges. Using sodium dodecyl sulfate (SDS) in denaturing polyacrylamide gel electrophoresis(PAGE) overcomes this problem because the SDS molecules bind to the protein chains in a constant ratio  of about 1.4g of SDS per gram of protein and overcome the charges of the ionizable side groups. This makes the net charge of all the proteins negative so that all the proteins migrate toward the positive anode when the electric current is applied.  SDS also changes the native conformation of the proteins into a constant rod-like conformation. This allows the protein chains to move according to their primary structure, and eliminates the differences in shapes that are found in the native secondary and tertiary structures. This allows separation of the proteins based predominantly on molecular mass.

  Alcohol dehydrogenase catalyzes the reaction of ethanol and NAD⁺ to form acetaldehyde and NADH. Since NADH is a product, absorbance values go up as the reaction progressed; therefore higher absorbance values mean an increase in activity. Lactose dehydrogenase catalyzes the reaction of pyruvate, NADH and H⁺ to lactate and NAD⁺. NADH is a reactant in this case; therefore lower absorbance values indicate an increase in activity. A progress curve plots absorbance values of NADH over the course of the reaction. The progress curve in an ADH experiment will show an upward trend while the progress curve in an LDH experiment will show a downward trend.

A method of protein purification is precipitation an anti-chaotropic salt. Ammonium sulfate can be used to precipitate out certain proteins of interest, such as LDH in protein mixtures. Ammonium sulfate should be added slowly when precipitating a protein because proteins precipitate out at different concentrations of ammonium sulfate. Ammonium sulfate decreases the amount of water available to the protein, causing proteins to precipitate out of solution.  Ammonium sulfate is used instead of sodium chloride because it is an anti-chaotropic salt, meaning that it favors protein folding rather than protein denaturation. Sodium chloride is moderately chaotropic and denatures proteins.

Activity assays are performed to observe how the activity (V₀) varies with different concentration of LDH. The enzyme Lactate Dehydrogenase (LDH) exists in two different forms. The H form is mainly found in cardiac muscle and it is optimized to catalyze the oxidation of lactate to pyruvate. The M form, found in skeletal muscle, is optimized to catalyze the reduction of pyruvate to lactate and the simultaneous oxidation of NADH to NAD⁺. This lab involves purifying the M form of LDH. Because NADH(a reactant) absorbs at 340 nm, measuring absorbance at 340 nm over the course of the reaction will allow us to determine the rate of activity(V₀) for LDH.

Blogging is so fun, I can write all kinds of pointless stuff that I enjoy and no one else cares to ever read. So I’ll take some time to share my knowledge of biochemistry, since I know the world is eagerly awaiting my contribution to the internet:

Affinity chromatography is used to purify mixtures of unknown proteins for a single protein of interest. The column matrix contains adenine monophosphate(AMP) immobilized on beaded agarose. The protein sample is diluted before running through the column to bring it down to a measurable range ( < 2 A₂₈₀). When the protein sample is run through the column, lactate dehydrogenase(LDH) binds to the AMP and becomes trapped in the column. The protein sample is followed by elution buffer to “wash the column” and elute out the contaminant proteins that have no affinity for AMP. Next a low concentration solution of nicotinamide adenine dinucelotide(NADH) is run through the column to elute out the proteins that do not bind strongly to the AMP. This “low NADH” is followed by a “high NADH” solution whose NADH concentration is high enough to elute out the LDH. Fraction collector tubes are used to collect a specified volume of solution as it elutes from the column. Measuring absorbance values of these collector tubes at A₂₈₀ allows us to detect the presence of aromatic amino acids, such as phenylalanine, tryptophan, and tyrosine and therefore estimate the amount of protein eluting from the column, following the “washes”.