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The study of the chemistry of living organisms, especially the structure and function of their chemical components, principally:Proteins,Carbohydrates, Lipids, and Nucleic acids. Biochemistry has advanced rapidly with the development, from the mid-20th century, of such techniques chromatography, X-ray diffaction, radioisotopic labelling, and electron microscopy. Using these techniques to separate and analyze biologically important molecules, the steps of the metabolic pathways in which they are involved (e.g. glycolysis and the Krebs cycle ) have been determined. This has provided knowledge of how organisms obtain and store energy, how they manufacture and degrade their biomolecules, how they sense and respond to their environment, and how all this information is stored and expressed in their genetic material. Biochemistry forms an important part of many other diciplines, especially physiology, nutrition, and genetics, and its discoveries have made a profound impact in medicine, agriculture, industry, and many other areas of human activity. (Reference "Concise Science Dictionary" Oxford New York OXFORD UNIVERSITY PRESS 1984, Modified Dr. P. Sieling UCLA School of Medicine 1996)
Dr. Sieling research involves the ability of the immune system to combat a particular bacteria known as staphylococcus aureus. The Staphylococcus aureus bacteria is responsible for extending the stay of hospital patients due to the infections caused by Staphylococcus aureus.
In one part of the experimental study of this bacteria and its associated immune response, Dr. Sieling grows the bacteria in a test tube. Next, he lyses the cell walls of the bacteria by adding an enzyme (lysostaphin). The cell walls of the bacteria, which are made up of sugars and amino-acids, are broken down by the enzyme leaving only the inner membrane of the bacteria . Due to the pressure existing within the cell, the membrane splits open dispersing the contents of the cell which include Nucleic acids RNA, DNA (molecules which contain its genetic code), proteins, lipids, and sugars.
The resulting solution is refered to as the lysate and is put into a centrifuge. The action of centrifigal force seperates the membrane particles from the solution (supernatant). This supernatant is then placed in two vials. One of the vials is treated with a protease which (degrades) produces denaturation of the proteins in the supernatant solution. The name of the protease is Pronase E and it is an enzyme secreted by a particular type of fugus. The reaction of the supernatant solution and the protease is allowed to take place over a period of three days.
After three days, a portion of the supernatent solution is tested with the protein assay that has been described earlier. This supernatant solution which has been treated with the protease should now have a concentration of approximately 10 micrograms of protein per milliliter (the proteins are not completely eliminated due to the equilibrium of the reaction of the protease with the protein, it never reaches completion). The supernatant solution that has not been treated with the protease should have a concentration of approximately 330 micrograms of protein per milliliter. It is important to know that the protein concentration in the supernatant solution, since we want to be clear about the fact that one of the supernatant solutions has protein and one does not.
The next step is to treat a sample of isolated peripheral blood (human blood of the mononuclear cell type) into two portions. One portion is treated with the supernatant solution that has been treated with the protease and has no protein. The other portion is treated with the supernatant solution that has not been treated with protease , this solution has all the proteins from the bacteria still in tact. These blood samples are then examined under a microscope after a period of four weeks to see if any T-cells have been stimulated by any of the antigens that were in the contents of the bacteria causing the T-cells to reproduce .
Typically it is known that the T-cells will be stimulated by Major Histocompatibility Complex (MHC) which is a molecule that is a protein within the Antigen Presenting Cell (APC). In a classical immune response reaction, a macrophage which is called an Antigen Presenting Cell consumes a foreign bacteria. The proteins from the bacteria are specific to that bacteria only. After the proteins are broken down into peptides the MHC molecule within the APC binds to the peptide and is tranferred to the suface of the APC where it presents its protein antigen. It is then recognized by a T-cell that has a specificT-cell receptor that will bond to the MHC molecule. This bonding that occurs and the co-stimulation of the T-cell by a dendritic cell cause the stimulation in the T-cell to produce interleukin-2 (IL-2), which is a growth factor for T-cells. This causes more T-cells to be produced that will have the same genetic information as that of the previous T-cell that was stimulated. All these T-cells will recognized the same specific antigen. By knowing this we can conclude that the supernatant solution that was not treated with the protease will stimulateT-cell growth.
Now through this type of experimental procedure it was discovered that there are APCs that present antigens that are non-proteins. The discovery of CD1 molecules which presents these non-protein antigens that are lipids (fats) and lipoglycan (lipo meaning fat glycan meaning sugar) is a recent breakthrough. This could lead to the creation of better vaccines to fight infections. So if the supernatant solution that was treated with protease (and therefore has no proteins) caused the stimulation of T-cells we can conclude that in fact non-protein antigens would be the cause of this stimulation of the T-cells since we knew there were no protein antigens to be detected in the supernatant solution that had been treated with the protease.
As I understand it, MHC molecules are different in each person only being slightly similar to that of close relatives, due to genetics. This makes vaccines that create MHC antigen stimulation of T-cells good for only a specific portion of the population that have genes that produce that specific MHC molecule type. On the other hand CD1 molecules are the same in everybody, so vaccines created to stimulate T-cells in this manner will cover a much larger portion of the population. And as I understand it Dr. Sieling along with some of the UCLA Administation Officials, are in the process of applying for a patent on this new type of procedure for creating vaccines. And if they do, Dr. Seiling might even be eligible to receive royalties for his part in the discovery. Way to go Pete. Look out for that Nobel Prize.
Dr. Sieling provided some technical notes about Protein Assays in general. The Bicinchoninic Acid (BCA) Method is one of the most recent advances in protein assay technology and is the one we will be using for determining the amount of protein in our solution.
First make up a solution in a test tube that is 2 mg of protein per ml. This is done by weighing 6.5 mg of Bovine Serum Albumin (BSA) (which is a known protein from cow blood that is simular to proteins found in humans) and mixing it with 3.25 ml of phosphate buffer saline (PBS).
Next take a seven serial microtubes and lable them in this order.
First tube- 2 mg/ml
Second tube - 1 mg/ml
third tube - 500 mg/ml
Forth tube - 250 mg/ml
Fifth tube - 125 mg/ml
Sixth tube - 62.5 mg/ml
With a pipet designed to extract solutions in volumes of microliters, place 25 ml of PBS solution into the second tube, third tube, forth tube, fifth tube, and sixth tube. Leaving the first tube empty.
Next place 50 ml of the BSA solution that is 2 mg of protein per ml in the first tube.Then in this order extract 25 ml from the first tube and place it in the second tube, extract 25ml from the second tube and place it in the third tube,
extract 25 ml from the third tube and place it in the forth tube,
extract 25 ml from the forth tube and place it in the fifth tube, and
extract 25 ml from the fifth tube and place it in the sixth tube.
This is a process known as serial dilution, in which the solution concentrations are systemacticly decreased by half. The solution concentrations should be in this order.
First tube - 2 mg of protein per ml
Second tube - 1 mg protein per ml
Third tube - 500 mg protein per ml
Forth tube - 250 mg protein per ml
Fifth tube - 125 mg protein per ml
Sixth tube - 62.5 mg protein per ml
Next in one row of a 96 well microtiter plate place 10 ml of each the standard solutions of known concentration into wells (one solution per well), in the seventh well place 10 ml of the unkown soloution. Duplicate this process in an additional row. You should now have 14 wells that have solutions.
Now from the protein assay kit, in a test tube, mix 6 ml of Reagent A (Bicinchoninic acid) with 0.12 ml of Reagent B ( Sulfur oxygen phosphide). Add 200 ml of this BCA reagent solution to each of the of the 14 wells that contain a solution.
Place the microplate in an oven set at 37° C. Allow 30 minutes for the reaction to take place. After 30 minutes take the microplate out of the oven.
Examine the microplate , in the row of wells that contain solutions of known concentration you should be able to see a color change in the solutions. The first well will have the darkest shade of purple due to the fact that it had the highest concentration of protein. The rest of the wells should decrease in shade and the last well with the lowest concentration will be practicly clear solution.
Examine the well which contains the unknown solution. You should be able to see a color change, which you can compare to the color changes in the row of known solutions. This can give you a rough estimate of the concentration of protein in the unknown solution.
For a much more acurate determination of protein concentration, the microplate can be placed in a special spectrophotometer which will measure the absorbance of light of the solutions in each of the well at 562 nm. The spectrophotometer is linked to a computer which allows the information of absorbance levels at 562 nm to be use in the spreadsheet program,"Microsoft Excel". A graph is created using the absorbance level on the Y-axis and the concentration of protein levels of known solutions on the X-axis.
Using the information points are plotted on the graph and a line is generated, then a slope is estimated for the line. Now we can determine the concentration of protein in our unknown solution by using the equation y = mx + b . Where y = the level of absorbancy at 562 nm , m = slope, b = the y intercept, and x = unknown concentration of protien.
I would like to especially thank Dr. Peter Sieling for providing ideas, information, materials for the protein assay, and for the tour of the laboratory where he conducts his research at the UCLA School of Medecine.
I would also like to thank Dr. James Murphy for his words of encoragement. It was fun and I learned more than I expected.
And last but not least I would like to thank my fellow chemistry club members for being a part and letting me be a part of the chemistry club these past semesters, Spring & Fall 1996.
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