Alcohol Dehydrogenase Protein: Histrical Background and Analysis

NB: We do not resell your papers. Upon ordering, we do an original paper exclusively for you.

NB: All your data is kept safe from the public.

Table of Contents

Introduction
A description of where the protein is found
The function of the protein in the organism
Properties of the protein
Cell lysis
Purification protocol
Assay and estimation of purity
Conclusion
References

Introduction

Alcohol dehydrogenases are a combination of enzymes which normally include several different types of such enzymes. For this family of enzymes the difference is caused due to their molecular structure. The difference in molecular structure makes them more efficient than others. These enzymes usually occur in various organisms in both animal and human bodies.The alcohol dehydrogenase usually acts as an alcohol breaker so as to enable proper digestion of alcohol by the body organs. There is a theory of evolution on this type of protein, where it is believed to have originated from formaldehyde dehydrogenase.

In 9137 the first alcohol dehiydrogenase was isolated from the bakers yeast and also its amino acid format and structure in dimension was also determined in the same year. This enzyme occurred due to the duplication of the main enzyme that is, ADH3 to ADHs.

A description of where the protein is found

There are five classes of alcohol dehydrogenase that are found in human beings, but the most profound one is the hepatic enzyme. The liver and the stomach is the most significant organ where these proteins are found. In this paper I am going to discuss more on alcohol dehydrogenase in the stomach. The main function of this enzyme is to oxidize ethanol in order to convert it into acetaldehyde.

Most of the alcohol dehydrogenases proteins are found in the chromosomes, where they metabolize various substances, including aliphatic alcohols, ethanol and retinol. It is necessary to note that these alcohols have a low boiling point and are always soluble in water.

Another thing to note is that, there are cofactors which are important in the oxidation process of alcohol in order to form a chain of hydrocarbons and thiols.There is also an amino acid cystein, which combines with the other groups of enzymes to help in the oxidation process of the alcohol. When units of amino acids come together they enable the protein to fold among each other, hence generating a disulphide bond.

The function of the protein in the organism

This protein belongs to a family of proteins which have aidehyde dehydrogenase properties; there are two main liver isoforms in this enzyme, which are mitochondrial and cytosolic. Eletrophoteric mobilities, kinetic energy and subcellular localization usually distinguish the properties of these two enzymes.

Alcohol is broken into acetaldehyde, and hence acetic acid radicals are finally formed, this occurs incase one consumes alcohol or eats foods containing alcohol. Through chemical reactions acetic radical mixes with coenzyme -A so as to form acetyl-CoA, which eventually goes to the Krebs cycle. Acetic acid, is then converted into acetyl-CoA by the enzyme ACSS2, which is found in the chromosomes. In order for this reaction to take place, the protein must act as a monomer, which then produces acetyl-CoA. Mitochondrial isozyme gene engulfs the mitochondrial isoform which is limited in Km for ethanol and is hence stationed in the mitochondrial matrix. The following chemical process shows how the ethanol is converted into acetaldehyde:

CH₃CH₂OH + NAD⁺ ’ CH₃CHO + NADH + H⁺

The most important thing this enzyme does is to break down the alcohol, which is contained in foods consumed by humans. It also enhances the consumption of alcohol. It is important to note that this protein takes part in the breakdown of other substances, such as ethyl glycol and methanol, which is oxidized to form formaldehyde and the former into glycolic acids.

Alcohol dehydrogenase works differently in many people according to their age, gender and the environment they are in; for example it is more efficient in young men compared to women of the same age group. There is another form of the dehydrogenase, which is the mitochondrial aldehyde dehydrogenase, which is described as a polymorphic enzyme. This enzyme is responsible for conversion of aldehydes into carboxylic acid through the process of oxidation.

The other form of alcohol dehydrogenase is the catalase proteins which further helps in breaking down of alcohol and through this process, hydrogen is formed. After the production of the hydrogen it is finally transformed into water, by the hydrogen molecules in the body.Catalase is profound in the brain, since it converts alcohol into water, although it is usually all over the body.

Alcohol dehydrogenase is usually efficient in cyclic and secondary alcohols but poorly in primary alcohols. It transfers hydride which is normally in the form of alkoxide ion into NAD⁺, which is later converted into NADH and Keton.This enzyme is a component of two zinc atoms, one which is the ligands and the other is the active one.

In bacteria and yeast this enzyme usually performs the work of fermentation, where pyruvate is transformed into acetaldehyde and carbon dioxide, hence being converted in to alcohol by ADH1, this normally the regeneration of NAD⁺.

There is also another family of these enzymes which usually contain iron in their bodies, these types of enzymes normally occur in bacteria. These enzymes mainly work with the help of oxygen molecules.

Properties of the protein

The table below shows some of the properties of this enzyme:

Property	Result
Size	Short chain
Soluble or membrane bound	Membrane bound
P1 of protein	high
Solubility in ammonia	soluble
pH	7.5
Sensitivity in metal ions	sensitive
Susceptibility	less
Requirements for cofactors	Yes

Cell lysis

Cell lysis is normally the disruption of the cell in order to get its contents. Some researchers usually lower the ionic strength of the cell so as to enable the cell to swell and then eventually bursting it¹². Some cells may pose some difficulties in the disruption process and it is important to put a surfactant in order to disassociate the components in the cells. There are different methods used to do this and they are as listed below:

Nitrogen burst method
Mechanical homogenizer
Ultra sound method with a probe

Where cells pose such difficulties in disruption the hypotonic method is normally used. Algae, yeast and bacteria are good examples of cells which have such characteristics. For these cells, it is usually difficult to disrupt such cells due to the presence of cell walls¹³. The stronger methods of cell lysis are as follows:

Enzymatic method; this method is usually used in cells involving subcellular isolation where there is preparation of protoplasts. Where this method is largely used it is usually expensive and irreproducible.
Bead method; this is the application of ultra sound on cells. This method generates radicals which normally react with molecules and hence making it disadvantageous.
Detergent method; this method is usually used to disturb lipids; it is good to determine the cell source type and the downstream application in order to choose this method.
Solvent use; this method is basically used for pathogenic and nonpathogenic bacteria cells. The use of this method usually denatures the cells.
Valve type processors; this method mostly forces the cell through a narrow valve with extreme pressure; these forces pull the cell apart, thus disrupting it.

After cell disruption, the cell is ready for centrifugation, this is a process which consists of a well refrigerated chamber which has a rotor that rotates at very high speeds approximately 100,000 r.p.m. In this chamber samples are placed in small cylindrical tubes and then placed to a rotating rotor. At these high speeds the rotor creates forces of around 800,000g to 1, 000,000g.Non uniform distribution of molecules is normally formed due to these speeds, but consequently sedimentation of molecules occurs too. Sedimentation of these molecules depends mostly on partial specific weight, shape and size of the molecules. Furthermore sedimentation of the molecules is dependent on the speed of the rotor and its size.

Purification protocol

This process of purification is done in order to separate the protein from other cells; this process starts with cell lysis in which the cell is disturbed and its components drawn to crude lysate solution. There after purification starts, through a method called ultracentrifugation. Purification process helps in breaking down protein cells into small and soluble components. Good examples of such soluble proteins are membrane lipids, cellular organelle and nucleic acid. Chromatography method is used to separate the proteins where the affinity of the molecule, its weight and the net charge are taken into consideration. Gel electrophoresis usually is used to monitor the process of purification where the isoelectric points and the molecular mass have been determined. More over, where the protein has a specific spectroscopic features one is in liberty to use spectroscopy, enzyme assays can also be used incase the protein has enzymatic activity.

In regard to the purification of natural proteins to be used for laboratory applications spontaneous steps of purification should be taken. Chemicals are added to the proteins while using genetic engineering procedure in order to make these kinds of proteins become easier to purify, and hence not disorganizing their physical structure and activity. After this process, a hestidine tag which consists of accurate amino acid sequence is used on the terminal of the protein. This outcome causes lysate to cross over the chromatography column, where nickel was earlier applied, but for the untagged specimens of lysate passes unnoticed.

Assay and estimation of purity

This assay is based on the absorbance shift of the dye coomassie, which occurs if the earlier red form coomassie reagent turns into blue, this happens due to the binding of the protein. Free electrons are donated to the ionized proteins blue part of the coomassie, hence causing the disturbance of the proteins and in this case the hydrophobic pockets are exposed. After the procedure is completed there is a non covalent attachment of the protein pockets to negative part of the dye, moreover the positive amine groups are coupled to the positive side of the dye. When the two interact in an ionic way they strengthen the bond. This self attachment of the proteins keeps the blue form of coomassie dye intact.

The absorption spectrum of the bond is 595nm.The chemicals in the protein cell usually make this procedure to be prone to interference. SDS usually causes assay interference in some detergents where there is high concentration. This is normally in two different modes of different concentrations. A good example of this is where an amicable binding occurs in an SDS below critical micelle concentration; this mostly protects the proteins from attaching themselves on other unwanted sites. Incases where the concentration is high above the normal critical micelle concentration the binding of the detergent usually occurs in the green part of the coomassie dye. This activity eventually causes a shift in equilibrium.

Conclusion

It is important to note that with the above procedure and experiment, I have been able to extract the protein and the properties of the protein have been well analyzed and proven. In this paper I have discussed the importance of alcocohol dehydrogenase in the orgasms and also its functions in the oxidation of alcohol.

This experiment concludes that alcohol dehydrogenase changes due to the overall number of amino acids present in the body but since the protein appears in all parts of the body it does not alter all its functions.Also sulphur is another element in the oxidation process.

References

Billinger, A. Gastric alcohol dehydrogenase activity in man: Alcohol and Alcoholism, Oxford University press, Oxford, 2002.

Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein: Utilizing the Principle of Protein-Dye Binding, Oxford University Press, New York, 2006

Danielsson, O. Enzymogenesis: classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line, Proc. Natl. Acad. Sci. U.S.A Journal. New Jersey, 2002, 89 (19): 924751 1409630.

Edenberg, H. Alcohol and aldehyde dehydrogenase genotypes and alcoholism in Chinese men, American Journal, Chicago, 2001, 48 (4): 67781 PMID 2014795.

Grekin, K. The development of alcohol use disorders: Annual Review of Clinical Psychology, Oxford University Press, New York, 2005.

Harper, R F. Illustrated Biochemistry, Oxford University Press, New York: 2006.

Hellgren, M. Enzymatic studies of alcohol dehydrogenase by a combination of in vitro and in silico methods, Ph.D. thesis, Sweden: Karolinska Institute, Stockholm 2009, p. 70.

Holde, V. Biochemistry, Effects of H2-receptor antagonists on gastric alcohol dehydrogenase activity: Digestive Diseases and Sciences, Harvard University Press, New York 2003, 36 (12): 16731679.

Kostof, S. Effects of H2-receptor antagonists on gastric alcohol dehydrogenase activity: Digestive Diseases and Sciences, 2nd edn, Oxford University Press, New York, 2005, p. 35.

Lehninger, R. Principles of Biochemistry, San Francisco Institute, San Francisco, 2005, pp. 180.

Minteer, SD. Microchip-based ethanol/oxygen biofuel cell: Lab on a Chip, Chicago University Press, Chicago, 2005, p.21825.

Molander, T. Ethanol absorption across human skin: Microdialysis Technique, San Francisco Institute, San Francisco, 2000.

Pastino, G. an Official Journal of the Society of Toxicology: Toxicological Sciences, Chicago University Press, Chicago, 2004.

Pozzato, C. The role of decreased gastric alcohol dehydrogenase activity and first-pass metabolism: High blood Alcohol Levels in Women. New England Journal of Medicine, London, 2006, 11; 322(2):95-9.

Shimokata, K. Mitochondrial ALDH2 deficiency: Oxidative Stress, Oxford University Press. New York, 2004.

Theorell, H. Mechanism of action of liver: Alcohol Dehydrogenase, Harvard University Press, New York, 2007.

Tooze, C. Introduction to Protein Structure, Garland Pub, New York 2009.

Click Here To Order Now!