Part 38

It is probable that these bodies do not exist as such in the fresh seeds in question but are produced therein from the other proteids by the alkali used in extraction. A further description of vegetable proteids will be found in the special paragraphs devoted to the study of these bodies in the principal cereals.

_Proteoses._—This name is applied to proteids which are not coagulated by heat, but most of them are precipitated by saturated solutions of neutral salts. They are also precipitated by nitric acid. They are formed from other proteids by the action of proteolytic ferments. The albumoses represent this group.

Protoalbumose is soluble in distilled water and weak saline solutions and is precipitated by mercuric chlorid and copper sulfate.

Heteroalbumose is insoluble in distilled water, but soluble in weak saline solutions, from which it separates when the salts are removed by dialysis. Deuteroalbumose is soluble in distilled water and saline solutions and is not precipitated on saturation with sodium chlorid. It is thrown out by mercuric chlorid but not by copper sulfate.

Vegetable proteoses are known as phytalbumoses, two of which have been found in the juice of the papaw mentioned above. They have also been found in cereals.

_Peptones._—These bodies are very soluble in water but are not thrown out by heat, by saturation with neutral salts, nor by nitric acid. They are completely precipitated by tannin and by strong alcohol.

The peptones are the only soluble proteids which are not precipitated by saturation with ammonium sulfate. The principal animal varieties are hemi- and anti-peptones. These forms of proteids do not appear to exist as such in vegetable products but are produced in large quantities by treating other proteids with pepsin or pancreatin. In sprouting plants, there appears to be a widely diffused ferment capable of converting the proteids of the cotyledons into peptonoid bodies and thus fitting them for entering the tissues of the new plant.

_Insoluble Proteids._—This class includes a miscellaneous collection of nitrogenous bodies not belonging to any of the definite groups already mentioned. Fibrin and gluten are types of these insoluble bodies. Fibrin is formed from the fibrinogen of fresh blood and causes coagulation. When washed free of red blood corpuscles it is a white elastic solid. It is insoluble in water and is converted into albumoses and peptones by trypsin and pepsin. It swells up when treated with a very weak one-tenth per cent solution of hydrochloric acid and dissolves to acid albumin when heated therewith.

Gluten is the most important of the insoluble vegetable proteids and forms the chief part of the nitrogenous constituents of wheat. It is readily prepared by washing wheat flour in cold water, as will be described further on. It is probably a composite body formed by the process of extraction from at least two proteid bodies existing in wheat. When dried it forms a horny elastic mass of a yellow-gray color. Gluten is composed of two bodies, one soluble the other insoluble in alcohol. The part insoluble in alcohol has been called vegetable fibrin, and the soluble part is subdivided into two portions, one unicedin or vegetable unicin, and the other glutin (gliadin) or vegetable gelatin. Gluten, according to some authorities, does not properly exist in wheat flour, but is formed therein by the action of water and certain ferments from free existing proteids. A better explanation of the composition of gluten is that of Osborne, which will be given further on.

=372. Albuminoids.=—In this paragraph the term albuminoids is not employed as synonymous with proteids but as characteristic of a class of bodies nearly resembling them, but, nevertheless, differing from them in many important particulars. Following is an abstract of their classification as given in Watt’s dictionary.[340]

_Collagen._—The nitrogenous portions of connective tissues are largely composed of collagen. By boiling water it is converted into gelatin. It may be prepared from tendons as follows: The tendinous tissues are shredded as finely as possible and extracted with cold water to remove the soluble proteids. Thereafter they are subjected for several days to the action of lime water, which dissolves the cement holding the fibers together. The residual insoluble matter is washed with water, weak acetic acid, and again with water. The residue is chiefly collagen, mixed, however, with some elastin and nuclein. With dilute acids and alkalies collagen swells up after the manner of fibrin. The organic nitrogenous matter of bone consists largely of collagen, which is sometimes called ossein.

_Gelatin._—When the white fibers of collagen, obtained as above, are subjected to the action of boiling water or of steam under pressure they dissolve and form gelatin. Isinglass is a gelatin made from the swimming bladder of the sturgeon or other fish. Glue is an impure gelatin obtained from hides and bones. Pure gelatin may be prepared from the commercial article by removing all soluble salts therefrom by treatment with cold water, dissolving in hot water and filtering into ninety per cent alcohol. The gelatin separates in the form of white filaments and these are removed and dried. Gelatin is insoluble in cold but soluble in hot water. It is insoluble in alcohol, ether and chloroform. Its hot aqueous solutions deflect the plane of polarized light to the left. Its gyrodynat varies with temperature and degree of dilution and is also influenced by acids and alkalies. At 30° it is [α]_{D}³⁰° = -130.

Gelatin is not precipitated by acetic acid nor lead acetate solution, in which respect it differs from chondrin.

If boiled for a day, or in a short time if heated to 140° in a sealed tube, gelatin loses its power of setting and is split up into two peptonoid bodies, semi-glutin and hemi-collin. Gelatin is easily digested but cannot take the place of other proteids in nutrition.

_Mucin._—This albuminoid, together with globulin, forms the principal part of connective tissue. It is also present in large quantities in mucus and is the chief lubricant of mucous membranes. It is extremely difficult to prepare mucin in a state of purity, and it is not certain that it has ever been accomplished. It is precipitated but not rendered subsequently insoluble by sodium chlorid, magnesium sulfate and alcohol. When boiled with sulfuric acid it yields leucin and tyrosin and, with caustic soda, pyrocatechin.

_Met- and Paralbumin._—Metalbumin is a form of mucin and differs from paralbumin by giving no precipitate when boiled. Both bodies yield reducing sugars when boiled with dilute sulfuric acid.

_Nuclein._—The nitrogenous matters which form the nuclei of the ultimate cells are called nuclein. Nuclein resembles mucin in many physical properties but contains phosphorus. It is also, like mucin, resistant to pepsin digestion. The nuclein of eggs and milk probably contains iron. Nuclein is found also in cells of vegetable origin and in yeast and mildew.

_Nucleoproteids._—These are bodies which yield both nuclein and albumin when boiled with water or treated with dilute acids or alkalies. Many nucleoproteids have the physical properties of mucus and the sliminess of the bile and of the synovial liquid is due to them. They are the chief nitrogenous constituent of all protoplasm.

_Chondrin._—Chondrin is obtained from cartilage by boiling with water. The solutions of chondrin set on cooling in the manner of gelatin. They are precipitated by the same reagents used for throwing out gelatin and mucin. Chondrin is also levorotatory. By some authorities chondrin is regarded as a mixture of gelatin and mucin.

_Elastin._—The elastic fibers of connective tissue are composed of this material. It can be prepared from the neck muscles by boiling with ether and alcohol to remove fats and then for a day and a half with water to extract the collagens. The residue is boiled with strong acetic acid and thereafter with strong soda until the fibers begin to smell. It is then treated with weak acetic acid and for a day with dilute hydrochloric acid. The acid is removed by washing with water and the residue is elastin. There is no solvent which acts on elastin without decomposing it. It is digested by both pepsin and trypsin with the formation of peptones.

_Keratin._—This nitrogenous substance is found chiefly in hairs, nails, and horns. It is essentially an alteration proteid product due to peripheral exposure. It is prepared by digesting the fine ground material successively with ether, alcohol, water and dilute acids. The residue is keratin. An imperfect aqueous solution may be secured by heating for a long time under pressure to 200°. It is also dissolved by boiling the materials mentioned above with alkalies, and when the solution thus obtained is treated with water, hydrogen sulfid is evolved, showing that the sulfur of the molecule is loosely combined.

Horn swells up when treated with dilute acetic acid and dissolves in the boiling glacial acid. When treated with hot dilute sulfuric acid it yields aspartic and volatile fat acids, leucin and tyrosin. Keratin, when burning, gives off a characteristic odor as is perceived in burning hair.

_Other Albuminoids._—Among the albuminoids of less importance may be mentioned neurokeratin found in the medullary sheath of nerve fibers; chitin occurring in the tissues of certain invertebrates; conchiolin, found in the shells of mussels and snails; spongin, occurring in sponges; fibroin forming silk and spiders webs; and hyalin or hyalogen found in edible birds’ nests.

The nitrogenous bases in flesh which are soluble in cold water, _viz._, kreatin, kreatinin, carnin, sarkin and xanthin are not classed among the albuminoid bodies, since they have a much higher percentage of nitrogen than is found in true proteid bodies, and are further differentiated from them by the absence of sulfur.

=373. Other Forms of Nitrogen.=—In addition to the proteids and albuminoids mentioned above, agricultural products may contain nitrogen in the form of ammonia, amid nitrogen and nitric acid. The quantities of nitrogen thus combined are not large but often of sufficient magnitude to demand special study. In general, these bodies belong to transition products, representing stages in the transfer of nitrogen from the simple to complex forms of combination, or the reverse.

For instance, the nitrogen which finally appears in the proteids of a plant has entered its organism chiefly as nitric acid, and the nitric acid which is found in a vegetable product is therefore a representative of the quantity of unabsorbed nitrogen present in the tissues at the moment when the vital activity of the plant is arrested. In some instances, it is found that the absorption of nitrates by vegetable tissues takes place in far larger quantities than is necessary for their nutrition, and in these cases the excess of nitrates accumulates, sometimes to a remarkable extent. In a case cited in the reports of the Kansas Agricultural Experiment Station, where Indian corn was grown on ground which had been used for a hog pen, the quantity of potassium nitrate found in the dried stalks was somewhat remarkable. When one of the stalks was cut in two and tapped lightly upon a table, crystals of potassium nitrate were easily obtained in the form of fine powder. On splitting the cornstalk the crystals in the pith could be seen without the aid of a microscope. On igniting a piece of the dried stalk it burned rapidly with deflagration. The percentage of potassium nitrate in the dried material was 18.8. Cattle eating this fodder were poisoned.[341]

In preserved meat products large quantities of oxidized nitrogen are often found, and these come from the use of potassium nitrate as a preserving and coloring agent. Ammonia is rarely found in vegetable tissues in greater quantities than mere traces, but may often exist in weighable amounts in animal products.

Amid nitrogen is found rather constantly associated with proteid matters in vegetable products. Asparagin and glutamin are instances of amid bodies of frequent occurrence. Betain and cholin are found in cottonseed.

The occurrence of nitrogen, in the form of alkaloids, is of interest to agricultural chemists in this country, chiefly from its presence as nicotin in tobacco and from a toxicological point of view, but in other localities the production of alkaloids, as for instance in opium, tea and coffee, is a staple agricultural industry. The methods of separating and determining these forms of nitrogen will be given further on. This description can evidently not include an extended compilation of the methods of separating and determining alkaloidal bodies, with the exception of those with which the agricultural analyst will be called upon frequently to deal, _viz._, nicotin and caffein and nitrogenous bases such as betain and cholin.

QUALITIVE TESTS FOR NITROGENOUS BODIES.

=374. Nitric Acid.=—Any nitric acid or nitrate which an agricultural product may contain may be leached out by treating the fine-ground material with cold water. From vegetable matters this extract is evaporated to a small bulk, filtered, if necessary, and tested for nitric acid by the usual treatment with ferrous sulfate and sulfuric acid. In the case of vegetable substances there will not usually be enough of organic matter to interfere with the delicacy of the reaction, but in animal extracts this may occur. Colored extracts should be decolorized with animal char (bone-black) before they are subjected to examination. It is not well to attempt to remove the organic matters, but, since they are more insoluble in water than the nitrates, the solution containing both may be evaporated to dryness and treated with a quantity of cold water insufficient for complete solution. The nitrates will be found in the solution obtained in a larger proportionate quantity than before.

=375. Amid Nitrogen.=—One or more atoms of the hydrogen in ammonia may be replaced by acid or basic bodies (alcohol radicles). In the former cases amids, in the latter amins result. In the ratio of displacement there are formed primary, secondary, and tertiary bodies determined by the number of hydrogen atoms replaced. The primary amids are the only ones of these bodies that are of interest in this connection.

The amids are easily decomposed, even on heating with water and the more readily with acids and alkalies, the amido radicle being converted into ammonia. A type of these reactions is given below.

CH₃.CO.NH₂ + H₂O = CH₂.CO.OH + H₃N.

On boiling an amid with hydrochloric acid, the ammonia is procured as chlorid whence it is easily expelled by heating with an alkali. In a body free of ammonia, an amid is easily detected by subjecting the substance containing it to the action of hot hydrochloric acid, filtering, neutralizing the free acid with sodium hydroxid, adding an excess thereof and distilling into an acid.[342] In case the quantity of ammonia produced is very small it may be detected by the nessler reagent.[343] Amids are soluble in a fresh, well washed preparation of cupric hydrate suspended in water. The hydrate also passes into solution forming a liquid of a deep blue color.

If amids be added to a cold solution of potassium nitrate in sulfuric acid free nitrogen is evolved.

=376. Ammoniacal Nitrogen.=—This combination of nitrogen may be detected by distilling the sample, or an aqueous extract thereof, with magnesia or barium carbonate. The ammonia is collected in an acid and detected therein by the usual qualitive reactions.

=377. Proteid Nitrogen.=—There are a few general qualitive reactions for proteid nitrogen and some special ones for distinct forms thereof. Below will be given a few of those reactions which are of most importance to the agricultural analyst:

_Conversion into Ammonia._—All proteid matters are converted into ammonia on boiling with strong sulfuric acid in presence of an oxygen carrier. Mercury is the substance usually selected to effect the transfer of the oxygen. Bodies which are found to be free of nitrates, ammonia and amids, are subjected directly to oxidation with sulfuric acid, and the ammonia produced thereby is distilled and detected in the manner already suggested. If nitrogen be present in the form of ammonia, amids and nitrates, the substance may be heated with an acid, hydrochloric or acetic, thrown on a filter, washed with hot dilute acid and the residue tested as above for proteid nitrogen.

_Biuret Reaction._—When proteid matter is dissolved in sulfuric acid, the solution, made alkaline with potassium hydroxid and treated with a few drops of a solution of copper sulfate, gives a violet coloration. This is commonly known as the biuret reaction, because the substance C₂H₆N₃O₂, biuret, left on heating urea to 160° gives the coloration noted in the conditions mentioned.

It has been found by Bigelow, in this laboratory, that if a solution is to be examined containing a very small amount of a proteid or similar body, the copper sulfate solution should not contain more than four grams of CuSO₄.5H₂O in 100 cubic centimeters of water, and the test should first be made by adding to the solution one or two drops of this copper sulfate solution, and then a strong excess of potassium or sodium hydroxid. The test may be repeated, using from one-half to two cubic centimeters of the copper sulfate solution, according to the amount of proteid present. If too much of the copper sulfate solution be employed its color may conceal that of the reaction.

Heating to the boiling point sometimes makes the violet color more distinct.

If a solid is to be examined it is first suspended in water, and in this state treated in the same manner as a solution. If solution is not complete, the mixture should be filtered when the color produced may be observed in the filtrate.

Proteoses and peptones give a red to red-violet and other proteids a violet to violet-blue coloration.

_Xanthoproteic Reaction._—Strong nitric acid produces a yellow coloration of proteid matter, which is intensified on warming. On treating the yellow mixture with ammonia in slight excess the color is changed to an orange or red tint.

=378. Qualitive Tests for Albumin.=—Albumin is one of the chief proteids and exists in both animal and vegetable substances. It is soluble in cold water and may therefore be separated from many of its nearly related bodies which are insoluble in that menstruum. In aqueous solutions its presence may be determined by the general reactions for proteid matters given above or by the following tests:

_Precipitation by Heat._—Albumin is coagulated by heat. Vegetable albumins become solid at about 65° and those of animal origin at a somewhat higher temperature (75°). Some forms of animal albumin, however, as for instance that contained in the serum, coagulate at a lower temperature.

_Precipitation by Acids._—Dilute acids also precipitate albumins especially with the aid of heat. Practically all the albumins are thrown out of solution by application of heat in the presence of dilute acids.

_Mercuric Salts._—Acid mercuric nitrate and a mixture of mercuric chlorid, potassium iodid and acetic acid completely precipitate all albuminous matters.[344]

The yellow or red color produced on heating albumin with the mercuric nitrate is known as Million’s reaction.

=379. Qualitive Test for Peptones and Albuminates.=—When peptones and albuminates are dissolved in an excess of glacial acetic acid and the solution treated with sulfuric acid a violet color is produced and also a faint fluorescence.

_Separation of Peptones and Albumoses._—In a solution of peptones and albumoses the latter may be precipitated by saturating the solution with finely powdered zinc or ammonium sulfate.

_Action of Phosphotungstic Acid._—All proteid matters in aqueous, alkaline or acid solutions, are precipitated by sodium phosphotungstate in a strongly acid solution. Acetic, phosphoric, or sulfuric acid may be used for producing the required acidity, preference being given to the latter.

_Action of Trichloracetic Acid._—In the precipitation of albumin by trichloracetic acid, there is formed a compound of the two bodies which to 100 parts of albumin has 26.8 parts of the trichloracetic acid.

The different albuminoid bodies obtained by precipitation behave in a similar manner. There are formed flocculent precipitates insoluble both in dilute and concentrated acids in the cold and also at a high temperature, with the exception of the hemialbumose compound.[345]

Albumin peptone, however, gives with the acid named in concentrated solution a precipitate easily soluble in an excess of the reagent. In the analysis of cow’s milk but not of human milk, this acid can be used for the estimation of the albuminoid substances. With both kinds of milk it can be used for the estimation of the albumin after the removal of the casein.

After precipitation of the albuminoid bodies, the milk sugar can be estimated by polarizing the filtrate and, volumetrically after removal of the excess of the acid by evaporation. By means of trichloracetic acid it is possible to separate albumin peptone from mucus and mucus peptone. A similar reaction is also produced by dichloracetic acid, but the reaction with this last agent is less delicate than with the other. Neither mucus nor albumin is precipitated by chloracetic acid.

=380. Action of Albumins on Polarized Light.=—Many of the albumins and albuminates, when in solution, strongly deflect the plane of polarized light to the left.[346]

The gyrodynats of some of the albumins and albuminates are given below:

Serum albumin [α]_{D} = -57°.3 to -64°.6. Egg albumin [α]_{D} = -35°.5 to -38°.1. Serum globulin [α]_{D} = -47°.8. Milk albumin [α]_{D} = -76°.0 to -91°.0.

Our knowledge of the gyrodynatic numbers of the proteids and allied bodies is too fragmentary to be of any great help in analytical work. In practice, the rotatory power of these bodies becomes a disturbing force in the determination of milk sugar.[347] A further study of this property of certain proteids may lead to analytical processes for their detection and determination, but no reliable methods for this can now be recorded.

=381. Alkaloidal Nitrogen.=—Only a general statement can be made here in respect of the detection of alkaloidal nitrogen in vegetable or animal tissues. Alkaloids are not found in healthy animal tissues and the description of methods for isolating and detecting ptomaines is foreign to the purpose of this work. In vegetable tissues the presence of alkaloids may be established by the following methods of examination.

The fine-ground tissues are made to pass a sieve of half millimeter mesh and when suspended in water are acidified with sulfuric. The mixture is then thoroughly extracted by shaking in a separatory funnel with petroleum ether, benzene and chloroform, successively. Some resins, glucosids and a few alkaloidal bodies not important here are extracted by this treatment.

The residue is made distinctly alkaline with ammonia and treated as above with the same solvents. In the solution obtained as last mentioned nearly all the alkaloidal bodies found in plants are contained.

All the alkaloids in a plant may be obtained by digesting the finely divided material with dilute sulfuric acid. The acid solution thus obtained is made nearly neutral with ammonia or magnesia, concentrated to a sirup, and gums, mucilage, etc. thrown out by adding about three volumes of ninety-five per cent alcohol. The alkaloids are found in the filtrate. The alcohol is evaporated from the filtrate and the residue tested for alkaloids by group reagents.[348] Potassium mercuric iodid and phosphotungstic and molybdic acids are types of these reagents.