This study was to examine and determine phylogenic relationships between various animals using electrophoresis results of their serum protein and Lactate Dehydrogenase isoenzyme activity. Using Bradford assays to normalize protein concentrations and gel electrophoresis for serum and LDH protein separations, it was determined that chicken serum was lacking in serum and LDH proteins while distinct bands were observed and noted for the mammalian serum with separate distrubtions of the LDH protein family in liver, thymus, heart, muscle, and serum. We concluded from the results that each animal sera displayed similarities in protein fingerprinting with chicken, horse, and rat being the outliers. Each tissue was also noted for its unique concentrations of each LDH subfamily Using our knowledge of the evolution of these species we found the evolutionary appearance of each protein.
The primary method of protein separation and analysis was native electrophoresis using agarose gels of 1.2% in Tris-glycine buffers of pH 8.6. Native gels measure both the size and charge of the proteins being studied, in this case those present in the animal serum and LDH in muscle and thymus tissue. (Steinmetz, et al. 2007).The proteins under study are not denatured and are used in their ‘native’ conformation as the name of the gel suggests.
Separation is achieved on the basis of size and electrical charge. The amino acid composition of a given protein determines the size and charge of the overall molecule and since the proteins under study are not denatured they fully retain their charges and chemical pH due to conformation shape. Under an electrical current the proteins will migrate to the anode and the distance traveled will be determined by the charge of the protein in the buffer solution. This will be heavily influenced by the pH of the buffer as higher pH will cause acidic proteins to be positively charges and vice versa (Wang, Jiou, et al 2007). Another influence in separation will be the size of the molecule in question. Larger proteins will be less motile than similarly charged smaller proteins, as the matrix of the gel will inhibit their movement down the lane. Small changes in protein size will have large consequences in separation(Abbott, et al 2006).
Staining involves binding Coomassie Blue dye to the protein. Coomassie Blue binds to the hydrophobic regions of a protein which is found on all proteins and thus is useful as a universal dye (Winkler et al. 2007). The more protein present in a region of the gel the more Coomassie Blue will appear. Once a gel has been stained with Coomassie Blue, alcohol and acetic acid are used to bind the protein coomassie blue compound in place while washing away excess dye, yielding the visible results of electrophoresis (Wang, Xuchu, et al. 2007).
Bradford assays are useful in obtaining a desired protein concentration and checking that concentration is correct. Coomassie Brilliant Blue is used to bind to the proteins and measured for the level of absorbency. If the solution is successful then absorbency should reach near 595 nm and a curve can be generated by plotting the absorbency versus the concentration of protein in the serum (Bradford 1976). This curve can be used to determine concentrations of proteins in unknown sera and to adjust it to a desired concentration. Using this one can create uniform concentrations of sera for electrophoresis, which in this lab was 5 mg/ml. This is vital for ensuring accurate and viable results during electrophoresis.
Serum is the plasma component of blood lacking clotting factors. Serum proteins can be broken into five distinct categories: Albumin, alpha 1, alpha 2, beta, and gamma globulins. Each has a designated purpose within the serum and is uniquely suited to that function. Albumin is usually used in the body as a transport mechanism for minerals, hormones, and nutrients. It also helps to maintain the proper balance of diffusion in the bloodstream plasma. Gamma globulins function as antibodies (Torpy, D. J. and Ho, J. T. 2007). Alpha 1, alpha 2, and beta globulins serve as transporters for lipids, proteins, and iron respectively (Agnello et al. 2007)(Yang, Kun and Sun, Yan 2007). The concentrations of each of these proteins is determined primarily by the species of animal and its current physiological condition, as sickness can alter the levels of proteins especially the antibody gamma globulins (Saleh, et al. 2007). Together these proteins constitute the majority of the proteins found in most mammalian serum.
Lactate Dehydrogenase is the main anaerobic energy enzyme in cells. It catalyzes pyruvic acid into lactic acid, releasing extra electrons to further break glucose down into ATP (Skory, et al. 2007). There are multiple subfamilies of LDH each of which is a separate isoenzyme of the basic protein, the primary ones being LDH 1 and LDH 5. LDH 5 is found in skeletal muscle and its conformation allows it to efficiently process lactic acid while LDH 1 is common to heart muscle and is not very efficient at anaerobic respiration. This reflects the needs of the muscles in which each resides: heart muscle must always pump blood for the survival of the organism as well as being close to arteries directly from the lungs and so does not rely upon anaerobic respiration, while the skeletal muscle frequently has to operate within the limits of oxygen capacity for exercise and uses LDH often. (Semenza, et al. 1996).
Protein fingerprinting allows one to identify proteins provided knowledge of the properties of said protein and conditions and rigging of the gel is set properly. This depends upon the pH of the buffer and the isoelectric point (pI) of the protein, as well as the gel. The isoelectric point is the pH at which the protein is electrically neutral and this is primarily determined by the buffer and how distant it is to the pI of the proteins under study (Pierre-Alain et al. 2006). The gel contributes in its porosity and thickness, a gel that is heavily porous will allow faster movement of larger proteins and thus will affect the results of protein fingerprinting (Hu, et al. 2004).
Phylogeny can be deduced from analysis of each completed electrophoresis gel. Phylogeny is the study and examination of evolutionary relationships between organisms. In our study this focused upon proteins, specifically serum proteins and LDH. Greater relatedness of the two species in question will be shown as greater similarity in protein expression and by extension of the amino acid sequence a genetic relationship as shown by electrophoretic separation of each serum (Blouin, et al. 2005). In addition, the presence or absence of a protein can also be utilized in this manner especially when considering the differences between classes and species of vertebrates (Levesque, et al. 2004). Accuracy is imperative as errors can lead to incorrect identification of phylogenic relationships (Teeling et al. 2005).
The first step in this lab was to conduct a Bradford Assay. Using varying concentrations of Bovine Serum Albumin (BSA), and a spectrometer, we were able to create a BSA curve to find an unknown samples protein concentration. In the next portion of the lab, we conducted an electrophoresis of various animal sera. We diluted various sera to the same concentration and ran them through an electrophoresis gel, in order to observe what levels of various proteins were present in each serum sample. The next part of the lab involved the electrophoresis of different lactate-dehydrogenase (LDH) isoenzymes alongside different animal sera. We ran two different LDH isoenzymes and various animal sera through an electrophoresis gel, to observe the varying content levels of the various LDH isoenzymes among the different animal species. The final component involved the electrophoresis of different LDH isoenzymes, but this time it was done so alongside samples from various tissues from the same animal. The LDH isoenzymes and the various tissue samples were run through a gel, to observe the different levels of the various isoenzymes among different tissues from the same animal.