We used electrophoresis to determine how "close" two species were. This involved learning about isoenzymes which are enzymes that function the same but have some different amino acids in non-active sites. We tested horse, cow, goat, and sheep serum for lactose dehydrogenase (LDH) isoenzyme. Goat and Sheep LDH isoenzymes are more closely to each other than to the cow or horse.
Electrophoresis is where organic molecules are placed in an electric field which displaces them. The displacement can be affected by weight, size, and charge of the organic molecule. We were using proteins in the plasma of Horse, Cow, Goats, and Sheep to find the isoenzymes of lactose dehydrogenase (LDH). The isoenzymes were targeted by a lactose sensitive dye. These enzymes should function the same, but the different lines should indicate where the enzyme has an altered amino acid in an non-active site. The differences or similarities of these isoenzymes should indicate the genetic closeness of species. It was hypothesized that Sheep and Goats would be a close match.
Using an agar gel in a buffer solution, we placed 15 microliters of timing dye and plasma in the wells. The plasma is the uppermost part of blood after being centrifuged. This contains the proteins that we will do our test on. Our power supply initially was set at 170 volts but this potential dropped during the three quarter hour electrophoresis time. After removal from the buffer solution, dying solution was added and the lines measured from the well. The dying solution contains the substrate of the enzyme which reacts and forms the products. These products trigger the dye. Thus we can isolate the enzyme and then see it. These measurements were limited by the thickness of the bands (3mm).
We measured, in mm, the distance from the well to the band of the isoenzyme. These bands varied in darkness which was qualitatively related in the sketch of the gel. The "top" side of the gel with the double cow wells was darker than the wells on the other side. Since the uppermost well was filled after the middle eight, decay cannot be the reason for the different darkness. A possible explanation should also explain the wider upper timing bands.
Two groups used the same gel. Our samples were gel slots 6 through 9. The two sets of samples show basically the same results. Upon analyzing another group's results, our readings differed from the other group by up to 7%. This can mostly be explained by the width of the bands which prevent measurements closer than 3mm. Furthermore, our horse sample lacked several isoenzymes from the other group. These differences do not, however, effect our final results that the cow and goat coincide.
The isoenzymes occurred consistently at about 30mm from the well. (Our timing bands were all at 42mm thus this direct comparison is possible). Only the horse sample did not have the 30mm band. The cow sample was the only one with a prominent middle band at 22mm. There was a progression of the lower band (15mm, 16mm, 17mm) from the Goats & Cows to Sheep, and then to Horses. (Question 1)
The derived "closeness" diagram concurs with figure 6-1. Goats and Sheep are really close with only one 1mm movement of an isoenzyme. Goats and Cattle are somewhat close with an addition of an isoenzyme. Horses are far from sheep with two movements of (or maybe completely different) isoenzymes. (Question 2)
An isoenzyme is an enzyme with some of the non-active site amino acids changed. These changes can be separated by electrophoresis because the protein can have a different polarized charge, weigh a different amount, or can vary in size. These factors affect its movement in the agar gel. (Question 3)
A human sample was not taken (Question 4).
The chemicals used in the stain had to isolate only the LDH enzymes. Thus there is a lactic acid. The other chemicals react to produces the brown colors so that we can see them. The chemicals used were probably chosen because of their availability and resistance to fluctuating laboratory conditions. (Question 5)
This technique can be used to determine if there is some rare mutation in the LDH enzyme that a researcher might want to track. The lack of an isoenzyme could also be used as an ID marker for genetically engineered cows and thus researcher can test for breeding patterns and their effect on the bovine populous. This technique could also be used to separate strains of virus protein shells. (Question 6)
I noticed a slight asymmetry in the dispersal pattern of the timing dye during the staining process. This probably won't affect the proteins, but it could signal that the dye may start diffusion too. We should just read the gel quickly. Faint lines might be better read after the gel sits for a while. (Question 7)
The data collected affirmed our hypothesis that Sheep and Goats were closely related relative to the other samples observed.