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Many techniques have been developed and improved for the purification of proteins till date. Purification allows us to isolate the protein in use thus enabling us to determine its structure. In this experiment, we purified the enzyme Alcohol Dehydrogenase (ADH) by Ethanol Precipitation and Metal Affinity Chromatography. After undertaking these purification methods, the purified protein from each experiment were subjected to analysis by SDS PAGE and Western Blotting. SDS PAGE allows us to verify the molecular mass of ADH and associate the weight with a sub atomic structure while Western Blotting allowed us to confirm that the protein was present in all samples. The best/effective purity obtained was by the ethanol precipitation method giving a purity of 1.600% with a purification factor of 5.820x insinuating a rather low purification. The ADH was also subjected to Mass Spectroscopy analysis which also verified the low presence
Alcohol Dehydrogenase (ADH), also known as aldehyde reductase is an enzyme pertaining to the highly conserved family of oxidoreductases found in many different organisms. The enzyme oxidizes alcohols to form aldehydes or ketones by reducing a NAD+ cofactor to NADH. This reaction is vital in many organisms including Homo sapiens to remove alcohols which are toxic to the organism. The reaction catalyzed is shown below:
CH3CH2OH + NAD+ ƒ CH3CHO + NADH + H+
The enzyme was first isolated from Saccharomyces cerevisiae in 1937 by Negelein and Wulff and the structure was first determined by X-Ray Crystallography. ADH was the first oligomeric enzyme to have its amino acid sequence and 3D structure determined. (2.3.4). It was later isolated from humans showing slight differences in structure but showed similarity in function. ADH is primarily found in liver cells in humans and is encoded by seven or more different genes. (1) Human ADH are broken down into 5 different classes ranging from I to V from which ADH Class I is the found mostly in the hepatic cells and is the most studied. ADH Class I is broken down into the α, β and γ subunits each encoded by the ADH1A, ADH1B and ADH1C respectively. Each subunit is found as a dimer of αα, ββ and γγ which coordinate with a zinc ion. (5) In human ADH, the zinc ion is liganded to the Cys 46, His 67 and Cys 174 residues with a possible 4th H2O molecule via hydrogen bonds which forms the active site. This coordination with Zinc is vital for enzyme stability and use.
Yeast ADH has 7 different isoenzymes. Each isoenzyme has a different gene which encodes for it and is named 1-7 for each gene. The genes encoded each isoenzymes are ranged from adh1p to adh7p. Yeast ADH has four polypeptide chains and nearly twice the molecular weight of human ADH. In Yeast, the principal ADH is ADH1 and is larger than the human ADH and consists of 4 subunits rather than the 2 in human ADH. The use of ADH1 is primarilary in anaerobic fermentation. ADH2 works in the same way but can be inhibited by glucose while ADH3 is only found in the mitochondria. (6)
Protein purification has become immensely important in the last century. The purpose of purifying a protein is to isolate it from a complex mixture of non proteins and proteins than to separate further from other proteins to be able to study the function, structure, enzymatic activity, isoform structures and interactions of the protein isolated.
The first purification technique for ADH purification relies on the precipitation of the protein by varying the concentration of Ethanol (or Ammonium sulphate). This is known as Ethanol/Ammonia Precipitation and is dependent upon the solubility of the protein at different concentrations of ethanol/ammonium sulphate. The theory behind this method states that the protein and ethanol solvent both compete for the free water molecules. As ethanol concentration is increased, the number of free water molecules reduces as the ethanol interacts with the water, thus encouraging more protein-protein interactions. This means less protein-water interactions leading to aggregation and precipitation of the protein. Using ammonium sulphate would also precipitate the protein but as the solvent used is a salt, the precipitation would be known as salting out.
The second purification technique used was Immobilised Metal Affinity Chromatography (IMAC). This technique metals are immobilised onto the stationary phase of the chromatography column to which the protein bind to. The most commonly used metal is Nickel though many of the transition metals can be used. Before running the ADH through the mobile phase, histidine tags are introduced to the open reading frame (ORF) of the protein (6x histidine) which bind to the nickel as ligands. This allows separation of ADH from other proteins which do not have the tags introduced. To elute this mixture, an analogue of histidine was added such as Imidazole. The Imidazole binds with greater affinity than the histidine thus causing the protein with the His tags to unbind and elute.
The purpose of the Ethanol Precipitation and IMAC was to isolate and purify the protein from S. Cerevisiae. The purified proteins were subjected to SDS-Page to examine the purity and Western Blotting to confirm purification. Once the purification was confirmed, the purity factor was determined by a MALDI-TOF procedure. This was followed by a trypsin digest and the results obtained were unconclusive. This could be a result of poor purification, contamination and/or lowered ADH activity due to purification procedures.
The last part of the experiment consisted of comparing the enzyme kinetics of Human ADH against a mutant isoform of ADH (ADH2), commonly originated in people with East Asian backgrounds. For both enzymes, the Km and Vmax were determined and contrasted by measuring the kinetics of the enzymes at different concentrations. Results proved that the wild type ADH showed higher Km and Vmax compared to the mutant.
Methods and Material
As stated in the RSM Manual.
The pure Yeast ADH extract showed a high specific activity of 568.92 Units/Mg. As a pure sample, the specific activity was predicted to be elevated.
'Purification- Steps taken'