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.