The precipitation reaction of protein, color reaction, sex reaction, charge and isoelectre determination.

related topics .First, the experimental purpose
1.
familiar with
precipitation reactions, color reactions and sexual reactions of proteins and proteins and their structure.
2. Master the measurement method and principle of protein and other electrical points.
3. Master the method of determining protein charge and its structure.II, experimental principle
(i) principle of precipitation reaction of proteins
The aqueous solution of proteins is a relatively stable hydrochloric collosome, because protein particles have many polar groups on the surface, such as -NH3 - COO-,—SH, - CONH2 and other water have a high degree of affinity, when proteins meet water, it is easy to be absorbed by proteins, protein particles outside the formation of a layer of water film (also known as hydration layer). The presence of water film separates protein particles from each other, and the particles do not collide and accumulate into large particles. Therefore, the protein is stable in the solution without precipitation. Another reason why proteins can form more stable hydro-collosomes is because protein particles carry the same charge in non-isoelectrogenic states, so that protein particles are mutually exclusive, maintain a certain distance, and do not condense and precipitate with each other.
protein forms a stable collome in aqueous solution due to its charge and water film. When certain physicochemical factors destroy the protein's water film or mediate the protein's charge, the protein collosal solution becomes unstable and precipitates. Proteins can be precipitated by adding the following
reagements
:
1. Salting such as ammonium sulfate, sodium sulfate, sodium chloride, etc. can destroy the water film around the protein colloid, while at the same time mediating the charge of protein
moor molecules
, so that the protein produces precipitation, this salting makes the protein precipitation out of the phenomenon called saline analysis. Salt analysis method is a common method of separating and preparing proteins, different protein salts need different salt concentrations, so adjust the salt concentration, can make the mixed protein solution in several protein segments, this method is called segmented salt analysis. However, neutral salts do not destroy the molecular structure and properties of proteins, so if the concentration of salts is removed or reduced, the protein dissolves again.
2. Plus
organic
solvents such as alcohol, acetone, etc. can make proteins produce precipitation, because these organic solvents and water have a strong role in destroying the protein molecules around the water film, so precipitation occurs. If the protein precipitation is separated from acetone or ethanol in time, the protein precipitation can be resolved in water.
3. Heavy metal salts such as mercury chloride, silver nitrate, lead acetate and iron triclosine. This is because proteins have negative ions in alkaline solutions that precipitate with positive ions of these heavy metals to produce non-soluble salts.
4. certain
acids
e.g. bitter acid, tylenic acid, tychloroacetic acid, etc.) can be sequestrated with protein salts that are not dissolved.
When acetone and ethanol were added to the protein precipitation by salt or low temperature, and the precipitation reaction using isoelectrogens, although the protein had precipitated and precipitated, its molecular internal structure did not change significantly, and the original biological activity was maintained. If precipitation is removed, the protein can be resoled in the original solvent. Therefore, this precipitation effect is called reversible precipitation. However, if organic solvents are added to precipitate isolated proteins at higher temperatures, or proteins that have been precipitated with organic solvents are not separated from organic solvents in a timely manner, the nature of proteins will change, which should be noted.
(ii) Principle of color reaction of proteins
Certain groups of protein molecules act with color agents to produce specific color reactions, different proteins contain
amino acids
are not exactly the same, color reactions are also different. Color reactions are not specific to proteins, and some non-protein substances can produce the same color reactions, so the results of color reactions alone cannot be used to determine whether the object being measured is a protein. Color reaction is the basis of some commonly used protein quantitative determination. Important color reactions are:
1.double peptide reaction
the urea
heating
to 180 degrees C, then the two molecules of urea shrink into a molecule of double shrinkure, and release a molecule of ammonia. In an alkaline solution, dichotomy can react with copper sulfate to produce a red-purple compound, which is called a double-shrink reaction. Protein molecules contain many peptide bonds similar to the structure of bi-shrink, so they can also react to form a red-purple complex. This reaction can usually be used to qualitatively identify proteins, but also according to the color produced by the reaction at 540nm color, quantitative determination of proteins.
2. The yellow reaction of protein
is a color-specific reaction of proteins containing aromatic amino acids, especially those containing tyrosine and tryptophan. Protein solution meets nitric acid, first produces white precipitation, heating then white precipitation turns yellow, and then add alkali color deepens orange yellow, because nitric acid will be the protein molecules in the benzene ring nitrification, resulting in yellow nitrobenzene derivatives. For example, thick nitric acid, such as skin, nails, and hair, turns yellow.
3. Mirren reaction
Mirren reagent is a mixture of mercury nitrate, mercury nitrite, nitric acid and nitric acid, protein solution added to Mirren reagents to produce white precipitation, heating precipitation turned red. Phenols
compounds
this reaction, tyrosine contains phenolic, so tyrosine and proteins containing tyrosine have this reaction.
4. tritone reaction
protein andtri ketone co-heating, the blue-purple reduction three ketones,tri ketones and ammonia acronym. This reaction is common to all amino acids α-amino acids. Other substances containing aminos also react.
5. Acetaldehyde acid reaction
adds acetaldehyde acid to the protein solution, and slowly injects thick sulfuric acid along the pipe wall, and purple rings appear between the two layers, a reaction that occurs in compounds containing pyridine. Tryptophan and proteins containing tryptophan have this reaction, and white gelatin without tryptophan does not.
6. The
arginine molecule contains a niobium-based molecule that produces red products in sodium hypochloride (or sodium subbrominate) and α-phenol in sodium hydroxide solutions. This reaction can be used to identify proteins containing arginine or to quantify arginine content.
.