Interpretation

If Petri Dish A, control:
A. Develops one or more precipitin lines:

1. It may be interpreted that the animal already had antibodies to one or more proteins in the "pure protein solution" that was added to the antigen well.
2. If precipitin lines are seen, their positions should be compared with those seen on plate B. In this case, it will be very difficult to determine purity using this method.

Any precipitin lines on plate B have meaning.

If Petri Dish B, test:
A. Exhibits precipitin lines

1. It is important to note the position of the lines and their congruity.
2. The interpretation of these lines is discussed below. B. Exhibits no precipitin lines
   1. Leave the plate a longer time and examine later.
   2. No evidence of the antigen antibody reaction could indicate antigen excess, try again with a lower concentration of the "pure" protein solution, or conversely it may indicate that the concentration is too low to be reactive. Review the concentrations that were used and alter the amounts or concentrations of antibody or antigen.

   The positions of precipitin lines on Plate B may be interpreted as follows:

   1. Complete identity
   
   When the precipitin lines from two antigen wells congruently fuse with one another, this is evidence of complete identity. Complete identity is shown (See diagram below) by the protein in wells 1 and 2, 2 and 3, 3 and 4, and 5 and 6.; not by protein in wells 4 and 5 and 6 and 1. It is noted that in some cases the same pattern may occur if two different proteins have the same diffusion characteristics and share common antigenic groups.
   2. Non-identity
   
   If the two antigens are different, their precipitin lines will intersect each other assuming the animal developed antibodies against whatever antigenic proteins were in the "pure" protein solution.In patterns of non-identity the intersecting lines continue to form "spurs". Non identity is shown by the precipitin pattern between wells 4 and 5.
   3. Pattern of partial identity
   
   If there are two proteins present that share a number of common antigenic groups and if the antiserum is capable of reacting with them in the concentrations used, partial identity is exhibited when the precipitin lines intersect but only one line continues past the intersection. See pattern between protein in wells 1 and 6. This pattern may be confused with one of non-identity.

   If only one line is seen formed between the antibody well and an antigen well regardless of concentration, but particularly in the higher concentrations, the solution of "pure" protein may be considered pure indeed. If there is more than one protein present, and the antibody production is maximal it may be assumed that the other protein would react with the antibody and also produce a precipitin line.

   

   6.0 Summary

   In order to ascertain that the isolated protein is pure, its study using specific activity, ion exchange chromatography, steric exclusion chromatography, electrophoresis with different buffers, isoelectric focusing, and reaction with specific antibody are all important. The methods should be based on different characteristics so that the distinction between similar molecules may be made. This discernment of a "contaminant" molecule is improved upon the use of a combination of at least four methods of separation and/or identification. The use of all of them greatly improves the detection of unwanted protein in the preparation.

   Methods To Assess Purity
   Practice Exercise 3.1:	Which of the following is true regarding methods to assess purity? No Response One boundary band on ultracentrifugation is proof of purity Immunochemical reaction is a sensitive way to assess purity One elution peak off one chromatographic column is a sensitive way to assess purity One stained band on a zone electrophoresis run is proof of purity

   Assessing Purity
   Practice Exercise 3.2:	Which of the following is true regarding methods to assess purity? No Response All of the methods discussed above are required to assess purity The more different methods that indicate one protein present, the greater the probability of purity Four of the methods described are enough to ascertain purity One stained band on two different electrophoreses at two pHs is proof of purity

   Immunochemical Assessment of Purity Practice Exercise 3.3:
   The immunochemical reaction shown in the diagram above has wells 1 through 6 which contain decreasing concentrations of a solution of protein that has gone through several purification steps. Well 7 contains antibodies to the protein solution. What do the precipitin lines indicate?	No Response The protein solution contains one pure protein One of the two precipitin lines between wells 1 and 7 are artifacts since both are not present at all wells. The most concentrated protein solution contains two different proteins None of the precipitin lines indicates identity.

   Lesson 3: Purity Assessment and Storage

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   Proteins - Analysis of Structure and Function

   B. Storage of Purified Protein Preparations

   Learning Objectives:
   Upon completing this topic and the practice exercises, the student should be able to:
   1. Identify common protein denaturing agents
   2. Identify the pH range tolerated by most proteins
   3. Relate a chemical agent with its denaturing qualities

   The stability of the protein that has been isolated and purified depends to some degree upon its molecular composition. Conditions for storing the protein must avoid denaturation, i.e. loss of secondary, tertiary, or quarternary structure. Our goal is to characterize the protein and it is important that its character not be altered by denaturation. Denaturing agents which cause the protein structure to be altered, also often alter the function.

   

   For example the molecular secondary structure of an enzyme, ribonuclease, depends upon its four disulfide bonds between its cysteine residues. A chemical agent, beta mercaptoethanol, reduces the disulfide bonds to sulfhydryl groups which destroys the secondary structure and also the catalytic activity of the enzyme.

   Some common denaturants are:

   1. heat
   2. pH conditions
   3. light and physical treatment
   4. chemical reagents

   Heat

   It is clear that the purified protein should be kept cool. Generally proteins in solution are maintained for 48 hours at 5^oC. Proteins when isolated and purified differ in their temperature tolerance from those which may be found at 37^oC in the body. Proteins that are to be extracted from blood serum require that the serum be separated from the red cells within 2 hours, especially when the red cells contain contaminants which will interfere with the serum extraction. Proteins from tisssues should be separated from the tissue mash as soon as possible to avoid proteolysis.

   Rarely but occassionally some proteins will lose their function upon refrigeration at 5^o. Examples of this are the lactate dehydrogenase isoenzymes # 4 and #5 (from liver and skeletal muscle) which lose their activity when stored at refrigerator temperature. These are customarily stored at room temperature for 2 to 3 days. Interstingly these isoenzymes may be stored for longer periods of time at 4^oC when their coenzyme, nicotine adeninine dinucleotide, or glutathione is added prior to storage.

   Denaturing Agents
   Practice Exercise 3.4:	Which of the following denature proteins? No Response Heat Chemical agents Extreme Acid or Alkali All of the above

   Most proteins do not maintain their structure when frozen and thawed and refrozen and thawed. It is advisable to store purified proteins in a freeze-dried state. This is accomplished by first freezing the protein in a flask with a vacuum fitting. The rapid freezing of the protein solution may be done using alcohol dry ice mixtures or liquid nitrogen. When the solution is frozen, the flask is attached to a vacuum and the frozen liquid is sublimated leaving a residue of pure protein behind. The pure solid protein is typically distributed among several vials, sealed and placed in a freezer at -20^oC.

   pH Conditions

   Most proteins are denatured by solutions at pH of less than 5 and greater than 9. This is particularly true of enzymes. When the pH of a protein solution is adjusted it is important to use a buffer system or to modify the pH by gently running mild acid or alkali down the side of the vessel with gentle mixing. The conformational changes that occur in proteins in very acid or alkaline solutions lead to loss of their function. There are some very exceptional proteins such as pepsin. It has a maximal conversion from pepsinogen at the pH of gastric juice, around 2.0.

   To characterize the protein it may be necessary to stress the conformation by the addition of sodium dodecyl sulfate or by a strong acid or alkaline environment. In these cases we are studying the conformational characteristics of the protein.

   pH Range For Protein Tolerance
   Practice Exercise 3.5:	At what pH range do most proteins retain their native characteristics? No Response pH 3 to 9 pH 3 to 8 pH 5 to 8 pH 5 to 9

   Light and Physical Treatment

   Protein solutions should be protected from light. Exposure to light may alter the conformation of proteins and particularly their affinities for several compounds. Examples are serum albumin and its affinity for bilirubin and that of hemoproteins for carbon monoxide. Ultraviolet light will over time denature the protein. For these reasons it is useful to store the protein in dark glass vials or in vials covered with aluminum foil.

   The handling of proteins in solution may also be a source of denaturation. Physical shearing may occur from harsh shaking or treatment.

   Chemical Agents

   Some of the chemical agents that affect proteins are:

   1. Oxygen and Thiol Oxdizing Agents
   2. Organic Compounds of Mercury or Arsenic
   3. Alkylating Agents
   4. Heavy Metals
   5. Organophosphate Compounds

   One of the agents that may be harmful to the purified protein or to proteins in solution is oxygen. This exposure may result in the oxidation of groups that are reduced in the native protein. For this reason the exposure of the protein or its solution to air is limited as much as possible. The most common source of error in physical treatment is the shaking of the protein solution to assure that the protein is solubilized. The protein is then spread on the surface of air bubbles and is easily denatured.

   Thiol groups of proteins are easily oxidized by such agents as oxidized glutathione and iodosobenzoate. The latter may also attack the methionine groups of proteins. Organic compounds of mercury or arsenic form mercaptides with thiol groups of proteins. Unlike alkylating agents the thiol interaction is reversed by the addition of an excess of a thiol such as cysteine. Among the alkylating agents, iodoacetate is well known. Chloroacetophenone and acrylates are others. Iodoacetate and other alkylating reagents react with thiol groups of proteins to give alkylated derivatives and hydrogen iodide. Heavy metal salts of silver, copper, mercury and leadare known to inactivate enzymes and will form complexes with other proteins. In the case of certain proteins and specifically enzymes, the use of ultra distilled water in their study is necessary to avoid the metal ions in ordinary water. The organophosphate compounds are known to inhibit enzymes which possess esterase activity, especially cholinesterase and acetylcholinesterase. These inhibitors are commonly known as "nerve gases" since their inhibitory affect results in loss of function by the central nervous system. They include compounds, Diisopropylphosphofluoridate(DFP),Tabun, Tetraethylpyrophosphate (TEPP), Paraoxon E 600, and Sarin.

   Although the agents described above are well known in their inhibitory effect on some enzymes, those which oxidize sulfhydryl groups and alkylate thiol groups will undoubtedly affect the conformation of and function of other proteins which contain such groups.

   Chemical Agents
   Practice Exercise 3.6:	Which of the following chemical agents will destroy disulfide bonds? Alkylating agents Organophosphate compounds Oxidizing agents No Response 1 and 2 only 2 and 3 only 1 and 3 only 1, 2 and 3

   Storage of Purified Protein
   Practice Exercise 3.7:	Which of the following is the best method for storage of purified protein? No Response freeze dried, in brown glass, stoppered vial, at -20 degrees C in solution at pH 7 for 1 month at 5 degrees C freeze dried at -5 degrees C in solution at a pH from 5 to 9, at 5 degrees

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   Final Instructions

   Press Button below for your score. After completing Lesson 3.0, including all practice exercises, press the "Submit..." button for Lesson 3.0 credit. After you press "Submit..."it is possible Netscape may tell you it is unable to connect because of unusually high system demands. If you receive no error message your submission you're OK. But, if Netscape gives you an error message after you press the "Submit..." button, wait a moment and resubmit or consult the attendant. End of Lesson 3.0 Purity Assessment and Storage of Purified Protein In Proteins-Analysis of Structure and Function Go To Lesson 4