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Protein Precipitation Reactions

Q.1-What is the difference between salting in and salting out?

Answer- A protein’s multiple acid base groups make its solubility properties dependent on the concentration of salts, the polarity of the solvent, the p H and the temperature. The solubility of a protein in aqueous solution is a sensitive function of the concentration of the dissolved salts. The solubility of a protein increases upon addition of low concentration of salt. This phenomenon is called salting in. The explanation for salting in is that as the salt concentration of the protein solution increases additional ions more effectively shield the protein molecules and thus promote protein water interactions, (protein -protein interactions are minimized as the like charges repel each other) resulting in increase in solubility of the proteins. 
At high concentration of salt, the solubility of the protein decreases. This effect is known as salting out and is primarily a result of the competition between added salt ions and protein molecules for the given solvent. At high salt concentration many of the added ions are solvated, so that the solvent available becomes insufficient to dissolve protein. Hence protein – protein interactions become stronger than protein water interactions and the protein is precipitate out. Salting out is the basis of one of the most common protein purification procedures. By adjusting the amount of salt concentration in a solution containing a mixture of proteins, the different proteins can be separated out.

Q.2- What is the difference between half saturation and full saturation?
Answer- Half saturation and full saturation signify the amount of salt required for precipitation of a protein. The amount of salt required for precipitation depends upon the molecular weight of a protein. There is generally an inverse relationship between amount of salt required and the molecular weight of the protein. The proteins with a large molecular weight need less salt. Globulins are precipitated by half saturation with ammonium sulphate, where as albumin with a low molecular weight is precipitated at full saturation. Fibrinogen is best precipitated by 1/5 th saturation with ammonium sulphate. The commonly used salt solutions are 
!) Ammonium sulphate  
ii) A mixture of sodium sulphate and sodium sulphite.
Q.3- Why is alcohol used as an antiseptic?

Answer- Water miscible organic solvents, such as Acetone and Ethanol are generally good protein precipitants because their lower dielectric constants lower the solvating power of their aqueous solutions for dissolved proteins. Hence the protein-protein interactions become stronger than protein solvent interactions and the proteins are precipitated out.

Alcohol rubbed over the injured site acts by precipitating membrane proteins of the micro-organisms and hence the multiplication of the micro-organisms is prevented at the site of tissue injury. Although alcohol cannot coagulate every single cell, it functions well to inhibit the growth and reproduction of many microorganisms, including bacteria, fungi, protozoa, and viruses. Human skin cells are more resistant to alcoholic coagulation than most microorganisms. This is why the human skin doesn’t coagulate if it comes into contact with alcohol.

Q.4- What is iso -electric p H precipitation?

Answer- Proteins generally bear numerous ionizable groups which have a variety of pK’s. At a p H characteristic of each protein, the positive charges on the molecule exactly balance the negative charges. At this Isoelectric point (p I)of the protein there is no net charge on it and the protein remains immobile in the electric field .The solubility of a protein is near a minimum near its p I. This phenomenon is the basis of a protein purification procedure known as isoelectric precipitation in which the pH of a protein mixture is adjusted to the pI of the protein to be isolated so as to selectively minimize its solubility. The precipitated protein is separated out from a mixture of proteins. Each protein has a specific Isoelectric pH (p I).

Q,5- What is the biochemical basis of curdling of milk ?

Answer- Curdling of milk is based on the principle of isoelectric pH precipitation. Addition of little amount of curd (Containing fermentation product of lactose in the form of lactic acid), lowers the pH of milk bringing it close to Isoelectric pH of Casein (milk protein). At this pH casein gets precipitated causing curdling of milk.

In normal conditions milk has a pH of about 6.5-6.7 and at this pH value the casein has a negative charge and there fore the casein micelles are relatively soluble, because they repel each others. Upon addition of curd ( lactic acid) casein acquires its Isoelectric point at 4.6 pH, that is at this pH value it has a quantity of positive charges equal to the quantity of negative charges and the positive part of each “micelle” is attracted by the negative part of the others, causing the formation of ionic bonds among the “micelle” working against the dipole-dipole bonds with water,i.e. protein-protein interactions get stronger than protein water interactions, so that the protein precipitates producing curdling of milk

Q.6- What is the benefit of using protein free filtrate for quantitative estimations?

Answer- Proteins present in the sample produce turbidity. The turbid solutions absorb more light-giving heightened optical density values. Since absorbance is proportional to the concentration of unknown substance. Hence the turbid solutions give falsely high levels of the substance present in the solution. Thus the proteins are eliminated from the solution before the quantitative procedures for the accuracy of the results.

Q.7- What is the principle of Heat coagulation test? What is the purpose of adding dilute acetic acid to the solution?

Answer-Heat causes denaturation of proteins by increasing the kinetic energy of the molecules and by breaking the weak bonds like electrostatic interactions, hydrogen bonds and hydrophobic interactions. The denatured protein becomes insoluble and precipitates out. Dilute acetic acid provides an acidic medium and brings the pH close to Isoelectric p H . At Isoelectric p H the protein has  a minimum solubility and is thus precipitated.
 In urinalysis, upon heating urine in an alkaline medium the phosphates produce the similar turbidity as observed with proteins but the turbidity disappears upon addition of acid if the phosphates are there but it persists if it was due to proteins.

Q.8-Why is milk or raw egg used as an antidote to heavy metal poisoning?

Answer- Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2,Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins.The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.
This reaction is used for its disinfectant properties in external applications. For example AgNO3 is used to prevent gonorrhea infections in the eyes of new-born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.
Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds.
This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body.

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