Molecular Components of Living Organisms

Assignment

Amber McElroy 5/29/2012

Molecular Components of Living Organisms

11337720

Amber McElroy

Molecular Components of Living Organisms Assignment

Introduction
Gel Electrophoresis is a method by which molecules are moved through a gel via an electric current for a set amount of time. Based on how far the molecules travel in relation to each other, assuming that some known molecules are used, molecules can be identified, and judgments made on their structure. The molecular weight of the molecule and the number of charged groups present effect how far the molecules move along the gel. Molecules with a low molecular weight will tend to move further than higher molecular weighted molecules, as they are smaller and encounter less resistance with the gel; however the charge of the molecule has more effect on the movement than the weight. A molecule with more than one charged group will likely move further than a molecule with one group, regardless of the molecular weight, this is due to the attraction of the charged groups to the flow of electrical current. There a many uses for gel electrophoresis, such as identifying unknown molecules, checking the purity of a molecule, DNA testing for example paternity tests, calculating molecular weight and a number more. For this report SDS-polyacrylamide gel electrophoresis is to be used to check the purity of lactate dehydrogenase, by comparison of the distance travelled to that of known molecules with a known molecule weight; from this the molecular weight of lactate dehydrogenase sample can be determined and compared to the literature value to determine the purity of the sample.

SDS-Polyacrylamide Gel Electrophoresis

The Gel

The type of electrophoresis that will be used in this assignment is SDS-Polyacrylamide gel or sodium dodecyl sulfate polyacrylamide gel, it is the most common electrophoresis gel used for separation of proteins. This is because it is beneficial for a number of reasons, such as the fact that it can withstand high temperatures and voltages, is a strong, transparent, mostly inert gel, it is also synthetic and depending on the amounts of certain chemicals it can have a range of different pore sizes; which is useful as the pore size or gradient can be changed the size to suit the type of proteins being analyzed. It is also able to be stained and de-stained without degrading the gel its self. The SDS- polyacrylamide gels are made up of several different components. These are Acrylamide polymers, Bisacrylamide, the pH buffer, a free radical catalyst and a stabilizer.

Molecular Components of Living Organisms

11337720

Amber McElroy

Acrylamide when dissolved in water undergoes auto polymerization; this is a slow process were acrylamide molecules join together into long single chained polymers. This does not cause the solution to form a gel; it does however become viscous as the polymers just tend to slide over each other. To form a gel bisacrylamide is added. This molecule bonds two of the acrylamide polymers together, so they are unable to slide over each other. However this is a slow process to overcome this there is a free radical catalyst added such as Ammonium persulfate; this initiates the polymerization. TEMED or Tetramethylethylenediamine is added as well as it is a free radical stabilizer, it also enhances the polymerization. Varying concentrations of both the acrylamide and the bisacrylamide effect the gradient of the gel; the smaller the gradient %, the smaller the pore size will be, therefore larger proteins will encounter more resistance. The buffer solution used in the making of the gel affects the overall pH of the gel; it can also affect the resolution of the gel. It is essential when choosing a buffer to not select a buffer that will react with or modify the proteins being run through the gel. The most commonly used buffers for this type of technique are Tris, Bis-Tris, or imidazole. In this assignment the buffer to be
used will be a Tris buffer. The Sample Preparation

The preparation of samples is important for this technique as the ingredients each play a different part, not only in modifying the protein, but in controlling the test. Samples are prepared in a solution of distilled water, a buffer (Tris HCL), glycerol or sucrose, 10% SDS, 2mercaptoethanol and saturated bromophenol blue. The buffer maintains the pH of the sample. The addition of glycerol or sucrose causes the sample to become denser, so that when the samples are loaded into the wells they will sink to the bottom of the wells. The saturated bromophenol blue is dye which allows the protein, which a normally clear, to be seen; it also enables the person running the test to see the distance travelled by the proteins, so that they know when to stop the experiment. The SDS chemical detergent, denatures the proteins, that have a secondary structure (tertiary structure as well as long as no sulfide bonds are present) so that they becomes a linear polypeptide chains and changes the overall charge of the protein to negative; because of this the proteins distance travelled through the gel is based solely on their molecular weight rather than the charge. As well as the SDS denaturation, the protein samples can also be heated with reducing reagents before been placed in the gel to denature them further. This normally involves breaking of the stronger sulfide bridges in proteins that have a tertiary or quaternary structure, allowing them to become linear polypeptide chains.
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Molecular Components of Living Organisms

11337720

Amber McElroy

The samples in buffer solution are then heated to about 95C for about 5 minutes; this heating period allows the SDS to denature and bind to the protein properly, as well as allowing the reducing reagent 2-mercaptoethanol to denature the protein completely as well.

(Electrophoresis, 2011) Electrophoresis is a relatively simple method and concept, it involves the movement of molecules through a gel matrix due to electrical current been passed through the matrix. Proteins in this case are either naturally charged or are chemically altered to give them a charge, in this case the proteins are given a negative charge. When the sample have been loaded and the buffer added to the top were the negative electrode is and in the bottom were the positive electrode is, an electrical current is passed through the gel from the negative electrode to the positive one; as the proteins are negatively charged they are repelled from the negative electrode and attracted to the positive one and they move with the flow of current down the gel matrix.

Method of Detection
The method of detection for this assignment is to be Coomassie Brilliant Blue R-250 or CBB; this is

due to the fact that it binds to proteins non-specifically. The stain is in a solution of methanol and acetic acid, the acetic acid fixes to proteins in the gel. It is necessary to use much more
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Molecular Components of Living Organisms

11337720

Amber McElroy

stain than gel as both the SDS and the CBB are anionic and this can affect the resolution of the gel. Once stained, the gel can easily then be unstained using the methanol and acetic acid solution without the CBB. The final result should appear to be a clear gel with blue banding were the proteins have separated to.

Results

Calibration Curve of the Molecular Weights of Proteins gained via Electophoresis

1000000

Molecular Weight (log)

10000

Series1 100 Linear (Series1)

1 0 20 40 Distance travelled (mm) 60 80 y = -1465.4x + 123188 R = 0.9665

Mr of Lactate dehydrogenase = -1465.4 x X +123188 =-1465.4 x 58+123188 =38194.8

Discussion
The calculated molecular weight of lactate dehydrogenase, was 38,194.8, which is much lower than that of the literature value which was 136,000. Lactate dehydrogenase has 5 different iso-enyzme forms, so would the sample still be considered pure if there are 5 different isomers present. Also each of these forms is made up if four subunits meaning the enzyme has a quaternary structure; when the sample was denatured these for subunits break apart and unfold making each of their molecular weight roughly a quarter of that of the un-denatured enzyme. This does not quite explain the molecular weight of the sample; if the sample molecular weight is multiplied by 4 it comes to 15277.6 which is a fair amount larger than that of the literature value. If this theory is assumed to be the correct one, it can be assumed that the sample is impure.

Molecular Components of Living Organisms

11337720

Amber McElroy

Bibliography
Electrophoresis. (2011, March 01). Retrieved May 31, 2012, from LABPLANET BLOG: http://blog.labplanet.com/2011/01/03/electrophoresis/ Lactate dehydrogenase. (2012, May 27 ). Retrieved May 29, 2012, from Wikipedia: http://en.wikipedia.org/wiki/Lactate_dehydrogenase SDS-PAGE. (2012, May 13). Retrieved May 29, 2012, from Wikipedia: http://en.wikipedia.org/wiki/SDSPAGE