Week 4: Protein Purification

Salting out This technique, known as "salting in" or "salting out", involves the gradual addition of ammonium sulfate to collect different fractions of precipitated protein Ammonium sulfate is a popular choice due to its high solubility in water, minimal susceptibility to temperature effects, and general compatibility with most proteins During this process, the hydrophobic groups on the proteinS become exposed to the atmosphere, leading to the attraction and aggregation of other protein hydrophobic groups The process, called salting out, entails adding a high concentration of an ionic species that disrupts the normal H-bond interactions between the solvent water molecules and hydrophilic resides on the surface of proteins . By gradually increasing the concentration of ammonium sulfate, contaminating proteins can be salted out first. Then, a higher concentration of ammonium sulfate is Used to precipitate the protein of interest, leaving yet other proteins still in solution Salting in Solubility Ammonium sulfate Ammonium sulfate precipitation precipitation Salt concentration Protein Purification Protein Puri cation The art and science of protein purification Most proteins are large molecules. They are smaller than DNA molecules, but they are tremendously large when compared to the molecules typical organic chemists are concerned with However, the process (disrupting cellular structure) has two side effects that may damage proteins: 1) cell disruption typically involves shearing forces and heat, both of which can damage proteins, and 2) cells normally contain proteases In most cells, proteases are carefully controlled; however, disruption of the cell usually also releases the proteases from their control systems, and may allow the cleavage of the protein of interest Property Technique Solubility Charge Hydrophobicity Size Function Stability Ammonium sulfate precipitation Ion-exchange chromatography Hydrophobic interaction chromatography Gel-filtration chromatography Affinity chromatography Heat treatment, pH treatment Overview of Lactate Overview of Lactate dehydrogenase dehydrogenase The full reaction that is catalyzed by LDH is: Lactate Dehydrogenase O CH C COO NADH. NAD CH OH CH COO During conditions in which pyruvate production from glycolysis exceeds the ability of the cell to metabolize the pyruvate, LDH converts the pyruvate to lactate, and thereby regenerates the oxidized NAD required for further glycolysis By convention, the above reaction proceeding from left to right is defined as the "forward" direction, but under the proper conditions, the enzyme can also work in the "reverse" direction There are two types of subunits, each coded by its own gene, with differences in amino acid sequence and properties: The LDHA gene encodes the M (muscle) form, predominant in skeletal muscle & liver The LDHB gene encodes the H (heart) form, predominant in heart muscle M and H subunits combine in differing ratios to form five isoforms, each composed of four subunits (H4, H3L1, H2L2, H1L3, L4), all with LDH activity but with different responses to allosteric effectors enzyme basics enzyme basics What is an enzyme? Enzymes are biological catalysts. A catalyst is a substance that participates in the reaction yet emerges at the end in its original form Enzymes act as a catalyst, but differ from ordinary chemical catalysts in that enzyme catalyzed reactions exhibit: Higher reaction rates Greater reaction specificity Milder reaction conditions Capacity for regulation Key characteristics Increase reaction rates Obey the laws of thermodynamics Catalyze the forward and backward reactions of reversible reactions Usually present in low concentrations because they are not consumed by reactions Are controlled via regulatory mechanisms Transition state of reacting substrates bound in enzyme active sites The chemistry of catalysis In a biochemical reaction, the reacting species must come together followed by electronic rearrangements (i.e. Some old bonds break and new bonds form) that result in the formation of products The point of highest energy is known as the transition state and can be considered a sort of intermediate between reactants and products The height of the activation energy barrier determines the rate of the reaction. The higher the activation energy, the less likely the reaction is to occur (or the slower it is) How do enzymes work? A catalyst enhances the rate of a chemical reaction but is not permanently altered by the reaction. Catalysts do this by lowering the activation energy required for a chemical reaction Enzymes help stabilize the transition state or in other words lowering the amount of energy needed to get the substrate into the transition state conformation The active site and enzyme specificity When a substrate interacts with the active site, its conformation and movement is restrained by its interactions with the active site The lock and key model suggests that enzymes bind to a single type of substrate because the active site and substrate have complementary structures In the induced fit model, noncovalent interactions between the enzyme and the substrate change the three dimensional shape of the active site, conforming the shape of the active site to the shape of the substrate in its transitional state conformation Importance of amino acid functional groups In addition to stabilizing the transition state, the functional groups in the active site play an important role in lowering the activation energy by providing an alternative lower energy reaction path Interaction of the substrate with functional groups also helps orient the reactants and bring them together correctly for the reaction to occur