Comparison of Muscle Proteins

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Comparison of Muscle Proteins to Infer Evolutionary Relationships of Mammals, Fish and Birds

Abstract
This experiment used electrophoresis to examine the makeup of muscle proteins from two mammals (cow, Bos taurus; bear, Ursus americanus), two aves (chicken, Gallus gallus; turkey, Meleagris gallopavo), and two fish (King Salmon, Oncorhynchus tshawytscha;  Albacore Tuna, Scombridae unclassified). The hypothesis was that as species have diverged evolutionarily, the makeup of their muscle proteins has also diverged. The further apart two species are evolutionarily, the less their muscle proteins will have in common.

The results did not support the hypothesis. There were not enough similarities between the fish proteins and not enough differences between the fish and the shrimp to reach a conclusion as to the commonality of their protein structures.

Introduction
Muscle tissue is composed of as many as 19 different proteins; the largest components are myosin and actin. All muscle tissue contains actin and myosin; these form the fibers that slide past each other as muscles contract and relax.  There are also a number of other proteins found in muscle tissue; these other proteins regulate movement, bundle and anchor actin and myosin along with others functions. As species have evolved, they have diverged in the quantities and types of these other proteins (Bio-Rad, 2006). The muscle proteins examined in this experiment were beef (Bos taurus), bear (Ursus americanus), chicken (Gallus gallus), turkey (Meleagris gallopavo), King Salmon (Oncorhynchus tshawytscha), Albacore Tuna (Scombridae unclassified) and shrimp (species unknown); included as an out-group. The hypothesis of this experiment was that as species diverged evolutionarily, the makeup of their muscle proteins also diverged. The further apart two species are evolutionarily, the less their muscle proteins have in common. If the hypothesis was correct, the two mammalian proteins would be similar, the two aves proteins would be similar, and the two fish proteins would be similar. Additionally, each of these three pairs of proteins would have more in common with each other than with the other pairs or with the shrimp protein.  The shrimp protein will have the least in common with the other proteins because the shrimp is an arthropod (protostome) and the others are all craniata (deuterostomia, chordata) (Freeman, 2011). Figure 1. Cladogram of hypothesis of the relationship among species and proteins.

This experiment used electrophoresis to examine the mix of muscle proteins in the seven species.  In electrophoresis the negatively charged proteins migrated through the gel toward the positively charged node. Since the larger proteins move more slowly than the smaller proteins, proteins separate out based on molecular mass. The molecular mass was determined by comparing the distance a protein traveled to the distance traveled by a protein of a known mass, contained in a maker, or standard (Bio-Rad, 2006)(McFarland, 2011).

Methods
The experiment was conducted in accordance with the Biology 212 Lab Manual (McFarland, et al, 2011) and the Bio-Rad Profiler Kit instructions (Bio-Rad, 2006). Proteins from the samples of raw muscle meat were extracted using Bio-Rad Laemmli sample buffer and denatured by heating at 95o C for five minutes. The proteins were then separated by electrophoresis at 200 volts for 25 minutes. A sample of myosin and actin and a kaleidoscope standard were included in the electrophoresis. A second gel was run to validate the results of the first gel. The contents of each gel well are shown in Table 1.

Table 1 Contents of Electrophoresis Gel lanes. Lane 1 was left blank. All samples were 10 microliters except the kaleidoscope standard sample was 5 microliters. Lane| 2| 3| 4| 5| 6| 7| 8| 9| 10|

| Kaleido-scope standard| Actin & Myosin standard| Beef| Bear| Chicken| Turkey| Salmon| Tuna| Shrimp|

The movement of each...