Bovine serum albumin is a model protein which has been conventionally used as protein standard and in many areas of biochemistry, pharmacology, medicine, etc. Radioiodination procedure for bovine serum albumin employing chloramine-T as an oxidant with slight modification was evaluated critically to establish the optimal conditions for the preparation of radiolabeled tracer (125I-BSA) with required specific activity without impairing the immunoreactivity and biological activity. Optimized radioiodination procedure involving 10 µg of chloramine-T along with 20 µg of sodium metabisulphite with 60 seconds incubation at 2oC yielded 125I-BSA with high integrity.
Key words: Bovine serum albumin, chloramine-T, radiolabeled, tracer
Serum albumin is the most abundant globular protein in the plasma and synthesized by the liver using dietary proteins. The physiochemical properties and structure of serum albumin is well studied and hence it serves as a model protein for several researchers [1-3]. Radioiodinated bovine serum albumin (BSA) is used in many areas of biochemistry, pharmacology and medicine.125I-BSA has been used conventionally in the tannin precipitation studies[4]. Hence, a well standardised procedure for the preparation of radioiodinated BSA could be useful for a variety of studies involving in-vitro quantification.
Although several methods for labeling 125I are available, radioiodination employing chloramine-T method is the most common method to introduce a radioactive atom into a large biological molecule [5]. The reaction starts with radioactive iodide anion, which is then oxidized using chloramine-T to a more reactive oxidation state. This reacts principally with aromatic groups in protein mainly phenolic tyrosine. The reac...
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...ubunit structure of the protein [8]. The damage caused due to radioiodination was experimentally proved by isoelectric focusing studies [9]. When conditions for reagent concentration was optimized, ideally for every one molecule of oxidant two molecules of reductant is needed to stop the reaction. The result was consistent with the previous radioiodination techniques studied for different protein molecules. It was demonstrated that when incubation time was increased it resulted in damage to the protein. The damage might have caused probably due to increased time of exposure of active iodide species with the protein. Studies on the effect of temperature gave yield of 75% when the reaction was performed at 2oC which is supported by the conclusion that at ambient temperature labeled BSA is in an aggregated form which lowers the iodination yield at low temperature [10].
...at keep organisms alive. “Proteins are the most structurally sophisticated molecules known” (Campbell, 1999) which is reason enough to study them. The techniques we learned in this lab form a basis from which a detailed study of proteins is possible. Following our procedure we were successfully able to set up a quantifying assay to determine the amount of protein within a milk sample, although our yield percentage was rather low. However, errors in this lab (in the form of a low yield percentage) may have an origin from our last lab. In the process of extracting proteins from the milk sample, we may have inadvertently lost some of the protein through erroneous measurements, or perhaps through poor handling of either ammonium sulfate or the dialysis tubing. While not sufficient enough (at this point) to invalidate our results, they do explain the major difference between the expected and the actual amount of protein extracted.
J.C. Biro, B. Benyó, C. Sansom, Á. Szlávecz, G. Fördös, T. Micsik, and Z. Benyó; A common periodic table of codons and amino acids. Biochemical and Biophysical Research Communications 306 (2003) 408–415.
To test for protein, the materials needed are a sample dish, Biuret's solution, and a dropper. Place a small food sample in a dish. Then proceed to add a dropper full of Biuret's solution, if protein is present, solution will turn from a clear to violet or blue. According to the tests, milk, and other milk products showed to have the most protein out of all the
...efore, is to detect and determine phenolic acids in pig blood by examining levels in red blood cells, plasma, albumin, low density lipoprotein (LDL),and very low density lipoprotein (VLDL). It is important to note that when a phenolic compound is present in body fluids, it generally occurs in one or several conjugated forms such as methyl, glucuronide and sulfate, (Proudfoot, Puddey, Beilin, Stocker, & Croft, 1997). There are several methods used to determine bioavailability of phenolic acids and possible metabolites in blood. Some of these methods include electron impact mass spectrometer (EI-MS), atmospheric pressure chemical ionization mass spectrometer (APCI-MS), electro-spray ionization mass spectrometer (ESI-MS), and matrix-assisted laser desorption/ionization mass spectrometer, (S Bertuglia, et al) as well as the more conventional HPLC after identification.
Albumin is a common protein in humans important for checking the health and detecting diseases. With a molecular weight of 65,000 and a density of 3.5-5.0 g/dL, it is made in the liver and released into the blood [1] [2]. Albumin has varieties of function. It maintains homeostasis to balance the amount of blood in the blood vessels [2] [4]. Albumin has a globular structure therefore it can form a colloid when mixed with water. Albumin is used for transporting drugs, lipids, and hormones by colloid osmotic pressure. Most colloid osmotic pressure comes from albumin [4]. Colloid osmotic pressure help to bind to both endogenous and exogenous substances. Drugs and other substances bind to albumin in the bloodstream so that the drug bound albumin can transport to the liver in order to make the drugs and substances less toxic to the target tissues and the water soluble substances. A lower count of albumin can result in a diseased state [4]. Albumin also can affect platelet system in the body. It can control blood clotting by binding arachidonic acid. This decrease production of thromboxane A2 and the activity of antithrombin increases and terminate clotting [1]. Blood clotting is important because it maintain permeability through vessels. Albumin is also important factor for metabolizing and detoxification drug and substances in our body [4].
Tymoczko, J. L., Berg, J. M., & Stryer, L. (2013). Biochemistry: A Short Course, 2nd Edition. New York, NY: W. H. Freeman and Co.
Due to the nature of amino acids, a titration curve can be employed to identify
Protein can be derived from two different food sources, these are animal proteins (meat) and plant proteins (vegetables, fruits, beans and nuts.) What a lot of people don’t know is that these proteins are not creates equal or share the same beneficial properties. Here I will discuss them both...
there are 2 types of proteins. Lean meats, eggs and dairy products are considered complete high quality sources...
= Before conducting the experiment I would conduct a simple test for the protein by placing a sample of the albumen into a test tube and add biurett reagent. This contains copper (II) sulphate and sodium hydroxide.
Analysis of a total of eighteen samples (six samples at each of the three test levels) which were prepared by adding known amounts of standardized chlorine solution to 0.1% sulfamic acid collecting solution.
Protein is one of the many things that can be seen on a nutrition label. “A protein is a linear sequence of amino acids linked together by peptide bonds…” (Food Proteins p.1). Amino acids make up a protein because they are connected by the peptide bonds. There are charged and uncharged amino acids as well as hydrophobic and hydrophilic amino acids. A charged amino acid is an amino acid “that can carry a charge depending on the pH” and an uncharged amino acid just doesn’t have a charge (Food Proteins p.1). A proteins structure can also be changed or denaturalized. “The native structure of a protein is energetic minimum under physiological protein. Any change in conformation away from this shape will represent an energy cost” (Food Proteins p.5). Proteins that are apart of our food and nutrition labels have the ability to be changed but energy needs to be used in order for the denaturalization of the protein to occur. There is a process known as the Udy Dye Binding Method that is used to analyze proteins. “In this procedure, ground grain is shaken with an orange dye solution. This acid dye forms an insoluble dye-protein complex with the basic amino acid building blocks of protein” (McDonald p.3). The dye used in this method is acidic and makes a protein that cannot be dissolved and has the building blocks of proteins still within it called amino
Some important AA sites within the protein sequence are: 431 – which is one amino acid in length, a proton donor and a binding site for thiamine pyrophosphate; 167 – which is one amino acid in length and a metal binding site; 197 – which is one amino acid in length and a binding site for, both, magnesium and thiamine pyrophosphate; 76 and 459 – which are, both, one amino acid in length and binding sites for thiamine pyrophosphate; 273 – which is one amino acid in length, a binding site for thiamine pyrophosphate and an essential site for catalytic activity; and 126 through 128 – which, altogether, comprise three amino acids in length and are the sites for nucleotide binding of thiamine pyrophosphate. The actual sequence’s chain begins at 2 and ends at 706 (Sakai et al, 1998); the first in the sequence (“1”) is removed as the methionine initiator. However, the “family tree”
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.
The relationship between a protein’s make up and it’s function is overly complex. Changing this relationship through the process of exposing the protein to heat at high temperatures over prolonged situations may put it at risk and may change the relationship. (Gerrard