{"id":1971,"date":"2024-03-03T17:59:19","date_gmt":"2024-03-03T17:59:19","guid":{"rendered":"https:\/\/cake.appscodestudio.com\/?p=1971"},"modified":"2024-03-03T17:59:20","modified_gmt":"2024-03-03T17:59:20","slug":"the-isolation-and-determination-of-proteins","status":"publish","type":"post","link":"https:\/\/cake.appscodestudio.com\/index.php\/2024\/03\/03\/the-isolation-and-determination-of-proteins\/","title":{"rendered":"The isolation and determination of proteins"},"content":{"rendered":"\n

Animal material usually contains large amounts of protein and lipids and small amounts of carbohydrate; in plants, the bulk of the dry matter is usually\u00a0carbohydrate. If it is necessary to determine the amount of protein in a mixture of animal foodstuffs, a sample is converted to ammonium salts by boiling with\u00a0sulf<\/a>uric acid\u00a0and a suitable inorganic\u00a0catalyst, such as copper sulfate (Kjeldahl method). The method is based on the assumption that proteins contain 16 percent\u00a0nitrogen, and that nonprotein nitrogen is present in very small amounts. The assumption is justified for most tissues from higher animals but not for\u00a0insects\u00a0and\u00a0crustaceans, in which a considerable portion of the body nitrogen is present in the form of\u00a0chitin, a carbohydrate. Large amounts of nonprotein nitrogen are also found in the sap of many plants. In such cases, the precise quantitative analyses are made after the proteins have been separated from other biological\u00a0compounds.<\/p>\n\n\n\n

Proteins are sensitive to heat, acids, bases, organic solvents, and\u00a0radiation\u00a0exposure; for this reason, the chemical methods employed to purify organic compounds cannot be applied to proteins. Salts and molecules of small size are removed from protein solutions by\u00a0dialysis\u2014i.e., by placing the\u00a0solution\u00a0into a sac of semipermeable material, such as\u00a0cellulose\u00a0or acetylcellulose, which will allow small molecules to pass through but not large protein molecules, and immersing the sac in\u00a0water\u00a0or a salt solution. Small molecules can also be removed either by passing the protein solution through a column of\u00a0resin\u00a0that adsorbs only the protein or by gel filtration. In\u00a0gel\u00a0filtration, the large protein molecules pass through the column, and the small molecules are adsorbed to the gel.<\/p>\n\n\n\n

Groups of proteins are separated from each other by salting out\u2014i.e., the stepwise addition of\u00a0sodium\u00a0sulfate or ammonium sulfate to a protein solution. Some proteins, called\u00a0globulins, become insoluble and\u00a0precipitate\u00a0when the solution is half-saturated with ammonium sulfate or when its sodium sulfate content exceeds about 12 percent. Other proteins, the albumins, can be precipitated from the supernatant solution (i.e., the solution remaining after a precipitation has taken place) by saturation with ammonium sulfate. Water-soluble proteins can be obtained in a dry state by\u00a0freeze-drying\u00a0(lyophilization), in which the protein solution is deep-frozen by lowering the temperature below \u221215 \u00b0C (5 \u00b0F) and removing the water; the protein is obtained as a dry powder.<\/p>\n\n\n\n

Most proteins are insoluble in boiling water and are denatured by it\u2014i.e., irreversibly converted into an insoluble material. Heat\u00a0denaturation\u00a0cannot be used with\u00a0connective tissue\u00a0because the principal structural protein,\u00a0collagen, is\u00a0converted\u00a0by boiling water into water-soluble\u00a0gelatin.More From BritannicaWhat Is the Difference Between a Peptide and a Protein?<\/p>\n\n\n\n

Fractionation (separation into components) of a mixture of proteins of different molecular weight can be accomplished by\u00a0gel filtration. The size of the proteins retained by the gel depends upon the properties of the gel. The proteins retained in the gel are removed from the column by solutions of a suitable concentration of salts and hydrogen ions.<\/p>\n\n\n\n

Many proteins were originally obtained in crystalline form, but crystallinity is not proof of purity; many crystalline protein preparations contain other substances. Various tests are used to determine whether a protein preparation contains only one protein. The purity of a protein solution can be determined by such techniques as\u00a0chromatography\u00a0and gel filtration. In addition, a solution of pure protein will yield one peak when spun in a\u00a0centrifuge\u00a0at very high speeds (ultracentrifugation) and will migrate as a single band in\u00a0electrophoresis\u00a0(migration of the protein in an electrical field). After these methods and others (such as\u00a0amino acid\u00a0analysis) indicate that the protein solution is pure, it can be considered so. Because chromatography, ultracentrifugation, and electrophoresis cannot be applied to insoluble proteins, little is known about them; they may be mixtures of many similar proteins.<\/p>\n\n\n\n

Very small (microheterogeneous) differences in some of the apparently pure proteins are known to occur. They are differences in the amino acid\u00a0composition\u00a0of otherwise identical proteins and are transmitted from generation to generation; i.e., they are genetically determined. For example, some humans have two hemoglobins,\u00a0hemoglobin\u00a0A and hemoglobin S, which differ in one amino acid at a specific site in the\u00a0molecule. In hemoglobin A the site is occupied by\u00a0glutamic acid\u00a0and in hemoglobin S by\u00a0valine. Refinement of the techniques of protein analysis has resulted in the discovery of other instances of microheterogeneity.<\/p>\n\n\n\n

The quantity of a pure protein can be determined by weighing or by measuring the ultraviolet absorbancy at 280 nanometres. The absorbency at 280 nanometres depends on the content of tyrosine and tryptophan in the protein. Sometimes the slightly less sensitive biuret reaction, a purple colour given by\u00a0alkaline\u00a0protein solutions upon the addition of copper sulfate, is used; its intensity depends only on the number of\u00a0peptide\u00a0bonds per gram, which is similar in all proteins.<\/p>\n","protected":false},"excerpt":{"rendered":"

Animal material usually contains large amounts of protein and lipids and small amounts of carbohydrate; in plants, the bulk of the dry matter is usually\u00a0carbohydrate. If it is necessary to determine the amount of protein in a mixture of animal foodstuffs, a sample is converted to ammonium salts by boiling with\u00a0sulfuric acid\u00a0and a suitable inorganic\u00a0catalyst, […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[],"class_list":["post-1971","post","type-post","status-publish","format-standard","hentry","category-proteins"],"Cooking_time":"","jetpack_featured_media_url":"","_links":{"self":[{"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/posts\/1971","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/comments?post=1971"}],"version-history":[{"count":1,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/posts\/1971\/revisions"}],"predecessor-version":[{"id":1972,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/posts\/1971\/revisions\/1972"}],"wp:attachment":[{"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/media?parent=1971"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/categories?post=1971"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/cake.appscodestudio.com\/index.php\/wp-json\/wp\/v2\/tags?post=1971"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}