BIOL 541 1st Edition Lecture 6 Outline of Last Lecture I. Characterization of Protein Outline of Current Lecture II.Edmund DegradationIII. Extract CrystallographyIV. Electron MicroscopeCurrent Lecture Biochemistry: Lecture 6: Carboxy peptidase enzyme technique, Extract crystallography.Sequencing from –COOH terminus: The most common technique is quick and dirty. Carboxy peptidase cleaves amino acid from –COOH terminus.Carboxy peptidase A- cleaves all amino acids except basic ones or proline.Carboxy peptidase B- cleaves lysine and proline.Carboxy peptidase C- cleaves proline.Method: The enzyme continues to nibble away on first and then second amino acid. Therefore, reaction condition has to be chosen in order to continue slowly.The sample is freeze dried followed by adding trichloroacetic acid to form precipitate. Then the amino acid is added on the analyzer. The graph shows that at 15 minutes, phenyl alanine is picked up, followed by serine coming out, thus this process is very messy.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.First carboxy peptidase A is added and lysine is cleaved. Then carboxy peptidase B is added to cleave 4-5 amino acids.Schlack- Kemp degradation: This is the chemical means of Edmund degradation. The reaction used does not allow for standards, and hence not used frequently. The organic extraction of amino acids following Edmund degradation has increasing loss of product as the peptide gets smaller and smaller as the product is partially extracted and washed out of the vessel.Mass spectroscopy: This method is used to determine molecular weight and sequence.A- The peptide bonds are ruptured using helium.B- Standard mass spectroscopy is done to determine the molecular weight.Disadvantage: This method can not distinguish identical amino acid molecular weight, such as leucine and isoleucine appear the same. Small peptides that are 25 amino acids long can be determined but Edmund degradation can perform on 40 amino acid length peptides.Advantages: It is not blocked by end termii that is seen in Edmund degradation. It is also faster than Edmund degradation.Extract crystallography: This is used for determining the 3D structure of the protein. To be visible, the wavelength ofthe particle must be smaller than the atom wavelength. In other words, x-rays can not be focused upon. Instead mathematical image is created since the analogy of microscope without the lens is inherent. Using the diffraction patterns 3D image is created.Disadvantages: It uses a lot of mathematical calculations. The proteins need to be crystallized in the same relative wavelength pattern and as such some proteins are not crystallizable. Further, small peptides crystallize forming different patterns. Also, larger proteins have more H bonds and hence fewer confirmations are seen. In some instances, the proteins are forced into abnormal confirmations.Nuclear magnetic resonance: Some nuclei have spin such as H, tridium, C-13. Some protein synthesis have culture with atoms, hence protein purification is done prior to subjecting it to magnetic resonance of varying strength. The nuclei flip at certain energy and is dependent on environment. The peaks are used to determine the 3D space and 3D structure. NMR in solution enables seeing protein in native confirmation with no artifact.Disadvantage: It is expensive with more math calculations and can be used for small peptides only.Electron Microscope: This equipment does not have the resolution to trace the peptide backbone but can trace the quaternary structure. The antibody structure has a light chain and a heavy chain that can be observed by this microscope. Proteases have been studied under this microscope as a dodecemer (12 subunits). There are 2 forms of it under the lens: a. hexagon and b. 2- layered structure.Hemoglobin:This is an extremely important component of the blood and transports oxygen. In obligate anaerobes, the oxygen is toxic and kills cells by forming free radicals. Small organisms receive oxygenby diffusion whereas the larger organisms need the circulatory system to transport oxygen. The solubility of oxygen in water is only 0.5% whereas in whole blood the value is 15%/ volume of blood. As more number of RBC are in the blood, the value of oxygen % volume in blood also increases. They are also termed as nano-machines because they not only pick up oxygen and deliver it but can also assess the body for the right amount of delivery.The binding curves for gases measure the partial pressure equivalence of gas concentration. The partial pressure value is denoted by calculating the tangent to the binding curve. Myoglobulin which is in the muscle has reserve stores of hemeoglobin to compensate for the increase in need during exercise. Hyperbolic curves denote the myoglobulin curve where 1 tangent denotes 1 binding affinitythat remains constant. Hemoglobin has a sigmoidal curve i.e. many curves. Hence the tangent denotes low affinity. Thereby the concentration increase in hemoglobin increases the binding affinityand increases the slope. Lungs have more concentration of oxygen in order to increase the affinity. On the other hand, tissues have low concentrations of oxygen and thus low affinity. As more activity is observed in cells, there is less partial pressure of oxygen, low affinity and thereby hemoglobin has an increase need to give up oxygen to the cells. Therefore the hemoglobin is tuned to the tissues’ needs for oxygen. Hemoglobin is a tetramer (quaternary structure) and myoglobulin is a monomer. Hemoglobin has 4 sub units where oxygen binding to 1 unit alters its conformation in other sub unit to increase the affinity. This is called as positive co- operativity. Bohr effect is seen when the curve shifts to the right as the pH drops.If oxygen is deficient, anaerobic respiration takes place by glycolysis and pyruvate forms. In such cases co- enzymes are used for pyruvate generation. The H is transferred to pyruvate to form lactic acid which explains the drop in pH. Therefore, hemoglobin releases more oxygen at low pH as low pH indicates low levels of oxygen and induces hemoglobin to release more oxygen. Hypoxia (chronic)is seen in high altitudes where more glycolysis releases more side