Partial Purification Of Amp Deaminase Biology Essay


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Reactions occurring in living cell require energy to react known as the activation energy and enzyme (biological catalyst) to speed up the reaction. AMP deaminase (AMPD), also known as AMP aminohyrolase is a hydrolase enzyme found in eukaryotes. Almost all processes of the cell require enzymes which are selective for their substrates, only specific substrates could fit into the active site of the enzyme to form products. The substrate that can fit to the active site of AMP deaminase, is AMP. Therefore like all enzymes, AMP deaminase is a protein which increases the rate of reaction by providing an alternative reaction pathway with lower activation energy (Adds et al, 2000). It catalyzes the deamination of AMP by acting upon its 5'-amino group to form IMP (inosine monophosphate) and ammonia. In addition, enzymes remain unchanged at the end of the reaction, similar is the case with AMP deaminase, it is not consumed in the reaction.

The role of AMP deaminase in the regulation of energy.

During exercise, muscles need high amount of energy to contract, therefore AMP deaminase is found in large amounts in the muscle which produces considerable amounts of ammonia from the deamination of AMP (Mahn et al, 1989). AMP concentration can be used to detect cell's energetic state; it is formed in the process of regenerating ATP from ADP during adenylate kinase reaction (2ADP ATP + AMP). The process takes place in the skeletal muscle to regulate energy. The muscle receives energy by the hydrolysis of ATP to ADP and phosphate. During exercise muscles require more oxygen to respire aerobically; due to low amount of oxygen present, muscles carry out anaerobic respiration which results in the producing of lactate. After exercise rapid breathing occurs to take in more oxygen for the conversion of lactate to pyruvate. Oxygen is the main component required to react with glucose to produce energy in the form of ATP. Therefore, when more ATP is required by the muscles during exercise, ADP (produced by the hydrolysis of ATP) is reconverted back to ATP to regulate energy delivery to muscles. (Adds et al, 2003). The equation (above) shows that two molecules of ADP are used to form one molecule of ATP and AMP. The reaction is catalyzed by the adenylate kinase enzyme which works together with AMP deaminase in the regulation of energy.

The role of the enzyme in the purine nucleotide cycle.

The enzyme (AMP deaminase) is part of a metabolic process that allows the conversion of sugar, fat, and protein in mitochondria into cellular energy by playing a major role in the purine nucleotide cycle and in the regulation of intracellular adenine nucleotides. The major metabolic role of the cycle is to produce ammonia that is used in the synthesis of glutamine (David J merkler, 1989). The catabolism of glutamine in the kidney leads to the production of ammonia which causes the neutralization of the tubular urine. One other main function of purine nucleotide cycle is to generate fumarate which in citric acid cycle is converted to oxaloacetate, to enable muscles to produce large amounts of ATP during exercise, similarly the cycle is significant for the conversion of adenine to guanine nucleotides to produce ammonia (for the production of energy). The process can occur successfully by increasing the amount of intermediates of tricarboxylic acid cycle and by regulating the level of AMP (Adds et al, 2003).

The function of the two enzymes: Adenylate kinase and AMP deaminase enzyme in the production of energy.

Both AMP deaminase and adenylate kinase work together to produce extra amount of ATP during exercise, which affects the adenine nucleotide pool (ADP+ATP+AMP) that returns to normal after exercise. This however occurs during the production or consumption of ATP. Enzymes such as ATPases (actomyosin ATPase in the muscle) produce and consume the same amount of ADP and ATP while in the adenylate and AMP deaminase system, two ADP is taken in to produce one ATP therefore this causes an imbalance in the ADP/ATP ratio. In addition, this leads to a decrease in the adenine nucleotide pool. The main role of the AMP deaminase enzyme is to increase the level of ATP/ADP ratio to balance the adenine nucleotide pool. This is achieved by decreasing the level of ATP production by the enzyme AMP deaminase through anaerobic glycolysis and by increasing the level of ADP and AMP (which activates glycolysis to produce energy). During Adenylate kinase reaction AMP formed, further breaks down IMP which activates the coupled reaction that converts ADP to ATP (Bernard karsonowiski, 2005). The production of energy (ATP) through this process prevents muscles form damaging during exercise and to slow muscle acidification. In addition, if the level of ATP is not regulated during exercise, the muscle becomes fatigue and results in AMP deaminase deficiency. More than 200 hundred patients have been affected by the disorder (doctor Manfred gross). The possible cause of the disease can be due to mutation in the AMPD1 gene which codes for the production of AMP deaminase and it cannot be diagnosed. There are various other possibilities that the enzyme (AMP deaminase) can be affected.

Factors affecting the activity of AMP deaminase.

Enzyme activity can be affected by the presence of other molecules such as inhibitors (molecules that attach themselves to the active site of the enzyme and affects enzyme activity). Effectors such as ADP, ATP, GTP, and pyrophosphate, allosterically inhibit the activity of AMP deaminase, whereas activators (are molecules that increase the activity of an enzyme) such as ATP, ADP, GTP activates the activity of AMP deaminase (Merkler,D.J., 1989). However, this has also been mentioned in a study that investigated the effect of effectors on the activity of AMP deaminase, the results revealed that GTP behaved as an inhibitor while ATP activated the enzyme (Ito et al, 1987). In addition, the same study mentioned the fact that studies on AMP deaminase of rat tissues showed that the enzyme occurred in three parental isozymes; A which was found in the skeletal muscle, B found in the liver and C found in the heart muscle. All the three isozymes of AMP deaminase showed different immunological and kinetic properties. However common feature is that they are enzymes and their activity can be affected by changes in temperature, pH and the concentration substrate used in a reaction. Enzymes require an optimum temperature and pH to work best, if a change occurs, the active site of the enzyme is affected. Similar to proteins, enzymes have long chains of amino acids that fold to form a three dimensional structure, they have a characteristic pH or temperature at which they function most efficiently, this is known as their optimum pH and temperature. Enzymes are denatured (the three dimensional structure of the protein is affected) if the temperature reaches above optimum temperature or if the pH changes (john adds et al check, 2000). Many enzymes have an optimum pH value at around 7, the optimum pH of AMP deaminase extracted from a lateral red muscle and dorsal white muscle of goldfish was between pH 6.8 and pH 7.0. At this pH the enzyme activity was high (Waarde,A.V, and Kesbeke,F., 2002). A study on the enzyme AMP deaminase from a Jumbo squid worked best at a temperature around 35 °C and denatured at a higher temperatures (R. Pacheco-Aguilar et al). On the other hand if the temperature increases the kinetic energy of the enzyme and substrate molecule increases, therefore this increases the rate of reaction (more enzyme substrate formation occurs) but if it increases above the optimum temperature, the enzyme activity is affected. The amount of substrate concentration also effects the rate of reaction (john adds et al check, 2000). If the amount of AMP (substrate) increases, the rate of reaction increases as more products are formed (IMP and ammonia). However, a study was carried out which showed that an increase in the concentration of AMP lowered the activation of the effector ATP, this might be due the fact that as the concentration of substrate (AMP) increases, it competes with other molecules (ATP) for the active site of the enzyme (AMP deaminase), therefore the enzyme is less affected by the effector. Similar is the case with an increase in the concentration of enzyme AMP deaminase, more active sites are available for the substrate AMP to join, and the speed of the reaction increases as the formation of products is rapid. This can be seen through enzyme assays which measures the rate of reaction.

Enzyme assays

Enzymes assays measure the concentration of substrate or product produced in a reaction to measure the rate of reaction. All enzymes including AMP deaminase can be measured through enzyme assays. To assay the activity of enzymes, changes in absorbance, fluorescence, pH, conductivity and other property differences of the substrates and products can be used. The rate of reaction of AMP deaminase can be achieved through spectrophotometric assays, which detects the change in the absorbance of light between reactants and products (between AMP and ammonia or IMP). Spectrophometers produce accurate results which makes spectrophotmetric assays more reliable. On the other hand a similar method that can be used is radiometric assays, which releases radioactivity that measures the amount of product produced over time. Both of the methods are useful but the disadvantage of using radiometric assays is that it only measures very low levels of enzyme activity. In an experiment, AMP deaminase was assayed using Spectrophotometric assays, the amount of ammonia produced in the deamination reaction of AMP was measured and the enzymes activity was determined from the absorbance at 625 nm (Ito et al, 1987).

Bradford protein assay

The Bradford protein assay is an example of an enzyme assay which measures the concentration of proteins in a solution. It is a type of procedure that relies on the absorbance shift which is initiated in the presence of a dye known as Coomassie. Proteins attach to the molecules of the dye and form a complex molecule. This is achieved by the two types of bond interaction between the molecules; the dye transfers its free electron to the protein which causes it to expose its hydrophobic pockets. This leads to the formation of vanderwaal forces of attraction between the hydrophobic pockets of the protein and the non-polar regions of the dye, which results in a colour change (from red to blue). The protein concentration is then measured using absorption spectrum, the amount of bound protein has an absorbance reading of 595nm. However, an increase in the absorbance from 595nm indicates the concentration of protein (Bradford, 1976).

Protein purification

Protein purification is a type of process that allows the isolation of a single type of protein from a complex mixture. The desired protein (AMP deaminase) is separated from a biological tissue, undergoing various steps in the purification process. The purpose of protein purification can be preparative, where large amounts of proteins are produced for subsequent use, it can also be analytical which produces small amount of proteins for research purposes. Searching for a particular type of protein maybe time consuming as in plants or animals, a particular type of protein is not distributed throughout the body, it might be found in different levels in certain organs or tissues. Therefore AMP deaminase as mentioned earlier is only found in eukaryotes it can be found in particular organs such as in rat muscle or chicken breast.

Extraction of AMP deaminase

Proteins such as enzymes can be extracted from a tissue by disrupting the tissue and releasing the enzyme into an aqueous extract. AMP deaminase can be extracted from a yeast cell (Merkler et al, 1989). Majority of cells show different characteristics, which needs to be considered during disintegration. This can be achieved through several methods. One of the useful methods is homogenization by high pressure which has always been the first method in protein purification. The next step that follows in the method is the centrifugation step which enables the soluble proteins present in the solvent to separate. After centrifugation a liquid layer is formed which contains the desired protein and it is trapped within the precipitated residue. One of the equipment most commonly used in breaking up of cell is the Waring blender (Scopes, 1994). An advantage of the method is that it also extracts proteases enzyme which breaks proteins in a solution. A study involved the same extraction method in extracting AMP deaminase from a mammalian (rabbit) myocardium using the homogenization followed by centrifugation technique. The left-ventricular myocardium of the male rabbits was rinsed with buffer and a 10% of the tissue suspension was formed when homogenized in a Waring blender (Thakkar et al, 1993). When the extraction of AMP deaminase occur, it is purified, therefore there are various techniques used in the purification of the enzyme.

Purification of AMP deaminase

Proteins maybe separated according to their molecular weight, polarity or their isoelectric points. An ion exchange column is used to purify proteins through their isoelectric points. The method is named as isoelectric focusing, the advantage of the method is that it allows the addition of several samples that make their way to the appropriate spots in the pH gradient. The use of an ampholyte concentration might be recommended when using the isoelectric focusing. This can lead to unreliable results, if proteins associate with it, a combined isoelectric point of both the protein and ampholyte components will occur rather the isoelectric point of the protein itself.


SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis is useful in protein purification by denaturing proteins with detergent sodium dodecyl sulphate (SDS) and running them through electrophoresis. Gel-electrophoresis is a method that establishes the movement of charged ions. Proteins are attached to the negatively charged detergent (SDS) which imparts a negative charge on the protein, this attachment denatures proteins by disrupting its non-covalent bonds. The negative charge formed by the protein and the detergent, represents the mass of the protein that is attached. The bound SDS produces electrostatic repulsion that causes proteins to separate in gel by unfolding and as they are negatively charged they move to the positive electrode (as unlike charges attract) in gel electrophoresis. SDS detergent is useful in gel-electrophoresis as it disrupts cell membranes and in isolation of the required protein from other proteins that are present in the same compartment. SDS is a molecule consisting of 12 carbon atoms (which are hyrdrophobic) attached to sulphate group. It has a structure similar to a fatty acid molecule but instead of a carboxylic acid it has sulphate attached (negatively charged). Sodium dodecyl dissolves fat and as the CH2 groups of the detergent are hydrophobic, it can dissolve proteins that are also hydrophobic. This is due to the membrane proteins being insoluble in water. One benefit of SDS -PAGE analysis is that insoluble particles when denatured are solubilised and are converted to single polypeptides, which is important as it avoids blocking the pores of native gels. However, proteins of identical sizes cannot be separated using this technique (Scopes, 1994). Migration of proteins occur through the differences in their sizes. Proteins are stained with silver stain to form bands on the gel which are eluted at the end of the process.

Figure 1.gel electrophoresis and the structure of SDS.

Salting out

Most enzymes in cell fluids are soluble proteins that are also soluble in salt conditions; this is due its polar interactions with aqueous solvent, ionic interaction with the salt and repulsive electrostatic forces between like charged molecules. Proteins are found to be less soluble at high salt concentrations known as salting out, as their solubility varies with salt concentration as well as with the ionic strength of the solution. Salting out is one of the most common, economical techniques used in enzyme purification, it uses neutral salts such as ammonium sulphate (NH4)2SO4), which causes proteins to precipitate (as ionic strength increases). Proteins have different composition of amino acids; therefore the salt concentration needed for a protein to precipitate out of the solution varies as well as they differ in solubilities at high ionic strength. The practical method causes the separation of proteins by altering the property of a solvent to form precipitate. The procedure involves dissolving of salt into the solution containing proteins (which consists of both hydrophilic and hydrophobic amino acids). In presence of a solution, the hydrophilic amino acids interact with water and form hydrogen bonds while the hydrophobic amino acids do not interact. The addition of salt affects the interaction of the hydrophilic amino acids with water and attracts water molecules towards it. Due to low level of water, proteins coagulate from each other by forming hydrophobic interactions with each other. The ammonium sulphate concentration is increased step wise and the precipitate protein is collected at each stage. However the precipitated protein of interest is recovered by centrifugation and by the addition of buffer. Ammonium sulphate is found to be one of the most effective salts as it consists of multiple charged anion such as sulphate, it does not affect enzymes and it is water soluble. The process relies mostly on hydrophobic interactions; other features can also affect solubility such as temperature or ph change. Solubility in salts is found to be high at pH 7, as at this pH proteins have the most charged groups, whereas temperature can cause an effect on the solubility of protein at high salt concentration, as it affects the hydrophobic interactions (Scopes, 1994).

Figure 2. Salting out


Centrifugation is a method that uses centrifugal force in separating particles of varying masses in a liquid. The process is followed by rotating the mixture containing proteins at high speed. A force produced during rotation, separates each particle according to its mass (as the force is proportional to the mass of the particle) during rotation. This results in the formation of a pellet (massive particles containing the required protein) and supernatant (non-compacted particles) in the test tube. Another type of ultracentrifugation is the Sucrose gradient centrifugation which produces sugar gradient (mainly of sucrose) this occurs due to the concentration difference of the mixture (highest concentration at the bottom and lowest concentration on top). A sample containing the required protein (AMP deaminase) is placed above the gradient and is spun at a high speed. Heavy macromolecules migrate towards the bottom during the process which is observed using a small observable window. At the end of the process proteins are separated.


Chromatography can also be used in protein purification. Many different chromatographic methods exist, whereas the basic procedure in chromatography is the flowing of solution (containing proteins) through a column consisting of various materials. Each type of proteins interacts differently with the materials of the column and they come out of the column when they separate, this is detected by their absorbance at 280 nm. The other type of chromatography that can be used in protein purification is size exclusion chromatography. The method uses porous gel to separate proteins in solution.

Ion exchange chromatography separates proteins by the degree of their ionic charge as proteins are found to have functional groups with positive and negative charges. In addition, anion exchange resins and cation exchange resins are used to separate positive and negative charges. Anion resins carries a positive charge and separates negatively charged compounds, whereas cation resins carries a negative charge and separates positively charged compounds. The method uses a column where buffer and the sample containing protein is added, buffer equilibrates opposing charged ions and competes with the protein to attach itself to the binding site of resins. Elution of charged compounds occur, weakly charged ions elute first followed by the elution of strongly charged ions. The analyte of interest can be detected through visible light absorbance. On the other hand Affinity chromatography can be used in protein purification.

Affinity chromatography is a technique that also uses resins which have ligands attached to it and which only separate specific compounds. This is shown in the lectin affinity chromatography, resins are modified with lectin and they attach to specific glycoprotein of a protein. Proteins that do not attach are eluted from the solution and a high concentration of sugar is added to separate the glycoproteins from resins to achieve protein of interest. They are then concentrated using several methods.

Proteins can be concentrated using Lyophilization technique; it follows the removal of volatile components and leaves the protein to dry. On the other hand, another useful method can also be used to achieve concentrated proteins, where the solution containing protein is pumped through a selectively permeable membrane (using high pressure) which also small molecules such as water to pass through while leaving larger molecules of protein behind.

In conclusion, AMP deaminase is a type of enzyme that regulates energy by carrying out the deamination of AMP as well as speeds up the reaction. Its activity can be affected if changes are made to the physical or chemical environment. It is only found in eukaryotes and can be isolated through homogenization as well as it can be partially purified through various techniques; such as Centrifugation, Affinity chromatography, salting out, Ion exchange, SDS PAGE electrophoresis.

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