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The nitrogen distribution in alfalfa hay cut at different stages of growth Clarke, Mills Forster 1937

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C U T A T D I T O K M T S T A G E S O F G B O W T H by M i l l s Forster Clarke ^ T h e s i s submitted for •the Degree of Master of Science i n Agriculture i n the Department of Agronomy. University of B r i t i s h Columbia A p r i l j 1937. TABLE OF CONTENTS Page INTRODUCTION I SCOPE OF THE IKVESTIGATION 1 PART I - REVIEW OF LITERATURE 2 A. Preparation of Plant Material for Analysis 2 Bi A n a l y t i c a l Methods 8 C. Discussion and Conclusion 25 D. The Method of Wasteneys and Borsook 27 PART I I - EXPERIMENTAL 31 A. Outline of Fie l d Experiment 31 B. Anal y t i c a l Methods 34 G» Experimental Results 41 33. Discussion and Conclusions 49 SUMMARY 77 ACK^OWLEDQEIISNTS 79 LITERATURE CITED 80 I THE .NITROGEN DISTRIBUTION.Iff ALFALFA HAY.. CUT, AT DIFFERENT STAGES OF GROWTH. INTRODUCTION. Notwithstanding the fact that quality and feeding value of hay crops i n general are dependent upon a large number of factors, each of which is important i n i t s e l f , protein content is perhaps most commonly used as a measure of general quality. This is p a r t i c u l a r l y true of a l f a l f a hay since i t i s notably high i n protein content as compared to the hays made from other types of forage plants. The use of protein content as the main c r i t e r i o n of feeding value i s undoubtedly j u s t i f i e d , since generally speak-ings a high content of digestible protein i s inseparably linked with the multitude of factors which c o l l e c t i v e l y are responsible for high quality. Furthermore protein i s required in large quantities i n the rations of a l l classes of livestock, hence due consideration must be given to the amounts of this essential and costly n u t r i t i v e constituent that may be present, when determining the value of any hay. The expression "crude protein" as usually applied to forage plants and their cured products, includes a l l the nitrogenous compounds present i n the material. Therefore, i t offers no information concerning the amount of nitrogen act-ually present as protein and the amounts present i n forms' representing different stages of protein synthesis and degrad-ation. I I Numerous experiments have shown that the cutting of hay • crops at different stages of growth together with the u t i l i z -ation of different methods of curing and storage have produced hays that differed markedly not only in t o t a l but also i n digestible protein content. The results of such experiments indicate that further information might be gained from a more detailed study of the actual nitrogen d i s t r i b u t i o n in hays produced under varying conditions. In addition the results of such a study would, in part, be indicative of the nitrogen metabolism of the plants from which the hay was made, since hay i s , i n r e a l i t y * plant mater-i a l with a portion of the water removed. However, owing to the multitude of chemical changes taking place during drying, t h i s material is by no means f u l l y representative of the o r i g i n a l plant. For this reason, any deductions that might be made concerning active nitrogen metabolism would have to be inter -preted i n a very broad and general fashion. SGOBB OE THE , INVESTIGATIOH It had been planned to make a more or less detailed study of the nitrogen metabolism of the a l f a l f a plant. How-ever, owing to a lack of f a c i l i t i e s for studying the plants in the green state, the problem was modified to permit of a detailed chemical study of the plant material i n a dried form. The investigation has been divided into two parts. The f i r s t i s a review of l i t e r a t u r e pertaining not only to the analyti c a l methods employed in the study of nitrogen meta-bolism in plants and plant products, but also to procedures concerned in.incomplete protein hydrolysates. Part I I of this paper i s devoted to the experimental phases of the problem; i t includes an outline of the f i e l d experiment together with f u l l details concerning the analy-t i c a l methods employed. The results obtained are discussed and conclusions drawn with respect to: ( l ) the nitrogen meta-bolism of the a l f a l f a plant at different stages of growth, (2.) the relationship between nitrogen d i s t r i b u t i o n and the quality of hay obtained from cuttings made at different stages of maturity, and (3) the s u i t a b i l i t y of the method of Wasteneys and Borsook for the study of the nitrogen d i s t r i b u t i o n i n plant material. PART I - REVIEW OE LITERATURE In the following review no attempt w i l l be made to cover completely the extensive l i t e r a t u r e dealing with nitrogen metabolism of plants. Discussion w i l l be confined to a con-sideration of the analytical methods commonly employed for the determination of the various nitrogenous constituents. Eor reasons to be discussed shortly, this w i l l of necessity include investigations dealing with the preparation of plant material for analysis. A. PRBPARATI OR OE PLANT . MATER IALs -The ana l y t i c a l d i f f i c u l t i e s encountered i n attempts to study the nitrogen metabolism of plants during the course of their entire growing and f r u i t i n g periods are many and varied. Consequently the results of different workers disagree to' such an extent that i t i s often d i f f i c u l t to draw significant or definite conclusions. The findings of numerous investigators have shown very cl e a r l y that the methods employed in the preparation of plant material are responsible for tremendous variations i n the analyti c a l results obtained. Hence i t is of no l i t t l e import-ance that the material be handled in such a manner as to insure no change i n the proportionate d i s t r i b u t i o n and identity of the nitrogenous compounds. Drying Material for Analysis - Prior to 1922 much of the work on the investigation of soluble and insoluble nitrogenous constituents mas carried out on dried plant material. Prev-iously, investigators had f a i l e d to appreciate•fully the effects of the methods of desiccation employed. Chibnall (1922) working with the common "bean (Phaseolis vulgaris, L.) ^observed that drying at r e l a t i v e l y low temperatures of 30° t o , 70° C. resulted in considerable proteolysis with consequent increase i n the simpler water-soluble nitrogenous products. These were, chie f l y ammonia (in the form of ammonium salts or as the amide nitrogen of asparagine) and mono-amino acids; the former being increased from 1 to 5 or 6 percent of the t o t a l leaf nitrogen, the amount of increase depending on the con-'s ditions of drying. Although the leaf proteins were found to be diminished i n amount they were not appreciably changed i n character. Tottingham et a l . (1924) and Link, and Schulz (1924) studied the optimum temperature at which autolytic changes in plant tissue were reduced to a minimum. They found that the greatest changes produced either by rapid drying or freez-ing resulted merely i n the coagulation of soluble proteins. Coagulation was found to be greatest at high temperatures, while at lower temperatures (32° to 40° C.) both coagulation -and proteolytic changes seemed to occur. G-reenhill (1933) investigated the effects of various drying temperatures upon the analysis of grass samples. Upon drying these i n shallow trays for forty-eight hours at a temperature of 40° C , he observed losses i n dry matter ranging up to 20 percent. When samples were dried 7 to 14 days, losses in dry matter as high as 12 percent were reported. At the same time, breakdown of "true protein" ranging up to 16 percent and 28 percent respectively for the two methods was noted, while increases i n the amount of pepsin hydrochloric acid soluble nitrogen up to 19 percent and 7 percent respectively occurred. The effects of drying on the nitrogen metabolism of barley leaves was studied by Richards and Templeman (1936), Samples of fresh leaves were extracted and analyzed immediate-l y after gathering, while duplicate samples were dried, sealed, and analyzed after a period of storage. Two types of hydro-l y t i c changes were observed to have occurred during desicca-tion J ( l ) a certain amount of amide gave rise to free ammonia, and (2) protein was hydrolyzed yielding amino acids. As regards the f i r s t type of hydrolytic change, the sum of ammo-nia and amide nitrogen was apparently unaffected by drying. In fresh leaf samples ammonia was found i n traces only, hence i t was assumed that the ammonia' i n the dried samples was derived from the amide i n the l i v i n g leaves* Richards and Templeraan consider the hydrolysis of a certain amount of protein, yielding amino nitrogen, as being much more serious than the hydrolysis of the amide nitrogen* The relative change i n protein content was found to be ne g l i -g i b l e , being of the order of 1 percent, but at the same time i t resulted in the small amount of amino nitrogen o r i g i n a l l y present being increased by approximately 15 percent. Thomas (1927) and Tottingham, Schulz and Leprovsky (1924) have demonstrated that rapid drying methods using temperatures i n the neighbourhood of 65° G. combined with effective aeration result i n a very slight alteration of the material "being analyzed. Many investigations have "been "based on a study of fresh material due to the fact that, unless extreme care i s exer-pised, changes in the proportionate d i s t r i b u t i o n of nitrogen in i t s various forms w i l l occur during the process of drying the plant tissue prior to extraction. Accordingly i n the work of Chibnall (1922), Vickery and Pucher (1931), Nightingale (1927), and Murneek (1926), fresh material is used exclusively. Extraction of Soluble Nitrogenous Constituents - The aim i n extracting plant material i s to obtain, i n as near to the o r i g i n a l form as possible, that portion of the nitrogenous material which is present i n a metabolically active form with-in the plant. In the pa.st there has been very l i t t l e agreement concerning the most effective method to employ for this purpose. Numerous methods have been proposed, a l l of which possess certain advantages and disadvantages. Extraction with hot alcohol i n concentrations of 50 to 80 percent has been recommended i n certain investigations (Tottingham et a l . 1927), for the reason that i t stops enzy-matic action quickly, precipitates some or a l l of the proteins, and i s a good solvent for the r e l a t i v e l y simple nitrogenous compounds. However, the precipitation of soluble proteins by alcohol during extraction is not considered as an advantage by a l l investigators. Soluble proteins are usually considered as being important constituents concerned i n plant metabolism and consequently methods which tend to preserve them intact are used. 1 Appleman and M i l l e r (1926) found that extracts of potato, tubers prepared by the use of hot alcohol yielded p r a c t i c a l l y the same amounts of non-protein nitrogen as did cold water extracts. Davidson, Clark and Shive (1934) show that hot water w i l l destroy enzymes about as rapidly as w i l l hot alcohol; also, hot water i s a s l i g h t l y better solvent for the simple nitrogenous constituents than i s alcohol. By the use of hot water, c e l l membranes are k i l l e d quickly and thereby rendered permeable to the soluble constituents. In the case of certain nitrogen determinations that can be made only on alcohol free samples, the use of hot water further s i m p l i f i e s the sub-sequent an a l y t i c a l procedure. Tottingham et a l , (1924) and Stuart and Appleman (1935) i n making aqueous extracts of soluble nitrogen, ground the plant tissues i n a mortar with the aid of sand. The chief disadvantage of such a method l i e s i n the fact that much labor and considerable time i s required. Moreover, Robinson (1930) has shown that i n the case of plants containing such unstable compounds as some of the cyanogenetic glucosides, the time required for the preparation of an extract by grinding permits of a loss of nitrogen or a change i n the d i s t r i b u t i o n of the nitrogenous fractions. Vickery and Pucher (1931), and Davidson et a l . (1934) have endeavoured to eliminate this alteration in nitrogen balance through shortening the period of extraction. This they claim to have e f f e c t i v e l y succeeded i n doing through b o i l i n g of the plant tissue i n water for a short period followed by moderate grinding i n a mortar without the aid of sand. Orcutt and Wilson (1936), on the other hand, point out that extraction of soluble material with either hot or cold water or alcohol i s objectionable because, in making a quantitative extraction, the f i n a l volume i s twenty times that of the o r i g i n a l material. Since actual amounts of the soluble nitrogen constituents i n plant materials are so very low, the further d i l u t i o n resulting from the previously mentioned methods of extraction increased the care required i n analysis. Concentration of the aqueous extract under diminished pressure i s sometimes suggested as a means of overcoming this d i f f i c u l t y . Aside from being extremely laborious, this operation leads to further error i n that i t tends to bring about decomposition such as aminolysis. In order to eliminate errors a r i s i n g from the use of solvents i n the preparation of extracts of soluble constitu-ents, numerous workers express the sap from fresh tissues by mechanical means. The addition of water is not required hence quantitative extraction is unnecessary. The anal y t i c a l results are expressed as concentration i n the sap, which may be con-verted to a dry matter basis i f desired. Varied applications .of the method are found in the work of Greathouse (1932), and Sayr and Morris (1931 and 1932). The usual procedure i s to express the sap through sub-jecting the material to a pressure of approximately 3000 pounds, following a preliminary grinding i n a b a l l m i l l . It i s claimed, that -when the material has "been previously ground in this manner,.the concentration of nitrogen does not vary throughout the extraction (Orcutt and Wilson, 1936). B.. iUJALYTICAL METHODS:-The methods employed in investigations r e l a t i v e to nitrogen metabolism i n plants are based primarily on methods that have been evolved at an e a r l i e r time for the study of "pure proteins". The study of pure proteins has been based on the assumption that the most direct way of obtaining an insight into the probable groupings which occur in the molecule of a complex substance is to break i t up into simpler ones whose constitution i s already known or may be determined with r e l a -tive ease. Various processes have been employed for breaking down the protein molecule, such for instance as acid hydrolysis, fusion with a l k a l i s , the action of enzymes or putrefactive bacteria, and oxidation* The application of this method has for a number of years resulted i n the accumulation of a large amount of r e l i a b l e data re l a t i v e to the composition of proteins both plant and animal. Further insight into the composition of proteins i s gained" from a study of the d i s t r i b u t i o n of nitrogen i n the molecule with a view to ascertaining whether i t i s present i n the form of mono- or di-amino acids. This separation depends on the fact that di-amino acids, by virtue of their strongly basic character, are precipitated from solution by the addition of phosphotungstic acid, whereas mono-amino acids are not. The substance being examined is f i r s t hydrolyzed by boiling with hydrochloric acid for several hours. The amount of amide and ammonia-nitrogen i s determined by d i s t i l l a t i o n with magnesia i n vacuo at 40° C* The di-araino acid nitrogen i s next determined by pre-c i p i t a t i n g the residue i n the fl a s k with an excess of phospho-tungstic acid and estimating the amount of nitrogen i n the precipitate by the Kjeldahl procedure. The nitrogen combined as mono-amino acids may be deter-mined d i r e c t l y i n the f i l t r a t e or by the difference between t o t a l nitrogen and the sum of the nitrogen fractions determined separately (Haas and H i l l , 1912). With regard to the study of plant proteins, the proteins of seeds alone have been thoroughly investigated. This i s easily understood since the seed proteins occur i n r e l a t i v e l y large amounts and can be isolated with comparative ease* As to the nature of the proteins and other nitrogenous compounds present i n a metabolically active condition within the growing plant there i s as yet very l i t t l e information available. The methods to be discussed shortly are concerned with the study of this material. Nitrogen Fractions Most Frequently Determined - The determinations made most frequently include: Total nitrogen, including n i t r a t e s , free ammonia-nitrogen, amide nitrogen, amino-acid nitrogen, and free nitrate-nitrogen. In some cases both mono- and di-amino acids, and occasionally soluble protein- and proteose-nitrogen are determined. The sum of the - 10 -various forms determined i s usually subtracted from the t o t a l soluble nitrogen and the difference called "Rest", "Residual", or "other" nitrogen. The amount of this f r a c t i o n varies con-siderably depending on the methods of analysis and as to i t s probable significance i n the physiological processes of the plant there i s l i t t l e information available. Figs. 1 to 5 inclusive i l l u s t r a t e graphically the procedures used by different workers i n an examination of plant material. With the exception of that of Mulay (Fig. 5 ) a l l the schemes agree quite closely with regard to the fractions determined and the order of procedure. Minor differences, however, do occur when one proceeds to the ultimate separation of the different fractions. This can be explained i n part through the fact that each procedure has been used on a different type of plant material. Owing to the variable composition of different plant species a procedure that can be used e f f e c t i v e l y on a l l types i s p r a c t i c a l l y impossible of development. „ Aside from the variations i n procedure, explanations for which have already been offered, other differences i n the methods occur. A l l schemes (excepting that of Mulay, Pig. 5 ) exhibit a certain amount of disagreement with respect to the vigor and type of hydrolysis which should be employed. Chibnall (Pig l ) hydrolyzes the water extract following the coagulation of soluble proteins with 4 percent hydrochloric acid for two hours. Thomas' (Pigs. 2 and 3) proposes two schemes of analysis - 11 -Fig. 1.scheme of analysis employed by Chibnall (1922) for studying the d i s t r i b u t i o n of nitrogen i n the leaves of the Runner, Bean. Fresh pLant tissue Residue Extraction with HgO< Solution Heated to 60°C. , cooled and f i l t e r e d Precipitate containing protein chlorophyll & colloids I washed with alcohol & extracted with ether to remove foreign material I Protein dried and weighed Clear f i l t r a t e 4 1/3" of f i l t r a t e boiled with 4% HCL for. 2 hrs. Cone, in vacuo-d i s t i l l e d i n vacuo over magnesia. (1) Amide H 4 N H 3 - N I Residue Remainder of f i l t r a t e P.T.Ai 'precipitation' Aliquot of f i l t r a t e ; sat. with ZnS04 (4)Proteose 2sT-Precipitate (2) Humin U F i l t r a t e Total; ff - determined on a i r dry sample by Xjeldahl method. Rest - Total IT minus sum of other H"de terms. Made alkaline to litmus with 30^ JTaQH-then just acid-cone. i n vacuo-made up to stand.voli (3) Mono-amino ¥ by procedure of Van Slyke l O O c c . d i s t i l l . 50cc* over magnesia treated (5)NH3-Jr with Devarda's alloy (6) NG3-¥ - 12 -Eig, 2. Scheme of analysis employed by Thomas (1927)for study-ing the nitrogenous metabolism of Pyrus malus, L. SCHEME I  Total N (Gunning Method) Extract with Hg,0 Total Soluble S" f (A) Insoluble £T Soluble Protein pptd. by c o l l o i d a l Ee(0H) 3 EiLtrate ( B ) 500 cc.. aliquot of f i l t r a t e treated according to (a). (a) 5oo cc. d i s t i l l e d i n vacuo with Mg(0H)2 Remainder of f i l t r a t e is hydrolyzed (according to (13) Remainder of f i l t r a t e ( B ) i s hydrolyzed with 20$ HC1 and d i s t i l l e d with ammonia (c) i s determined on the d i s t i l l a t e .. +. n 1Ca(0H) 2 d i s t i l l a t e ^ residue 1 I Residue (d) f i l t e r -ed from "melanin" U(e) and freer (mono-& di-amino U(f) i s determined on the f i l t r a t e .(D) after cone, i n vacuo d i s t i l l a t e (g) Residue (h) contains free cdnc.& f i l t . •ammonia (c) & from Ca(0H)2 free and com- "Melanin" & bined Amide Jff(j) humin N(lc) Eree & com-bined mono- & di-amino K(l) is det. on the f i l t r a t e E after cone.in vacuo* H( l ) - N(f) 58 combined mono- and di-amino IsT. - 13 -Fig. 3. Scheme of analysis employed by Thomas (1927) for study-ing the nitrogenous of Pyrus malus, L. SCHEME II The whole of the non-protein U f i l t r a t e (B) i s taken and hydro-lyzed with A% HC1 and d i s t i l l e d with Ca(0H)2 Residue D i s t i l l a t e (a) contains free ammonia and asparagine N i s Residue f i l t e r e d from the Ca(0H) 2 "melanin''^ and humin U(d) F i l t r a t e (c) treated, after cone, with pho sphotungstic acid f i l t r a t e residue (d) Free mono-& di-amiho IT i s det* on the f i l t r a t e . • ( e ) Residue con-tains the basic H compounds. - 14 -Pig. 4* Scheme of analysis proposed by Orcutt and Wilson (1936) for the study of nitrogen, metabolism i n the soybean plant. Fresh plant tissue . Hydraulic press Residue Juice I Heated to 70°C. , cooled .and f i l t e r e d . Chlorophyll, c o l l o i d s , coagulated proteins. 2 - 4cc. I 1.Total 3ST. 2.Amide-ir+-Mi3-H" Clear f i l t r a t e locc i 2cc. of enzyme solution, incubated at 40°C12 hrs I 20% laHS0 3 i n steamer for 3 hrs. I S O 2 removed by H 2 S O 4 and: aeration. I Made alkaline & aerated. — r A c i d i f i e d with acetic acid,diluted to 25cc. 5cc. 3.a-amino H" 4.Total Basic H 3. minus 5» *• Basic Amino. K 4. minus Basic Amino N B Basic non-amino N 20cc. 3? • T • JL» precipitation. Neutralized, then a c i d i f i e d with acetic acid, and diluted to SOcc* lOcc. ! 5.a-amino H" 20cc. ! 6.N03-N - 15 -Pig. 5. Scheme of analysis employed by Mulay (1931) for the determination of t o t a l , soluble, soluble protein, non-protein, and insoluble nitrogen* Total Nitrogen (Gunning Method) Extract Mat s r i a l with H&O Total Soluble Nitrogen (det. on aliquot of extract) Soluble Protein Nitrogen pptd. by c o l l o i d a l J?e(OH)3 § pH 4. Soluble Non-Protein Nitrogen determined on f i l t r a t e from Pe(0H) 3 pption. Insoluble Nitrogen determ. on residue from HgO, extraction Total Sol. N - Sol. Non-Protein N = Sol. Protein N Insoluble N Sol. Protein N " T o t a l Protein N. 6.25 x protein N = Proteins - 16 -that d i f f e r with respect to the vigor of hydrolysis. In the f i r s t procedure (Fig. 2) a portion of the extract previously freed of proteins i s hydrolyzed with 20 percent hydrochloric acid for 12 hours. In the second procedure (Fig. .3) the whole of the non-protein nitrogen f i l t r a t e i s hydrolyzed with 4 percent hydrochloric acid* The difference i n the vigor of hydrolysis results i n a variation with respect to the d i s t r i b -ution of the mono-amino and the di-amino acids, and the sub-stances determined as amide nitrogen. Most of the complex peptide linkings would be decomposed by the stronger hydrolysi: hence the amide' nitrogen of this procedure includes the com-bined as well as the free -CO-KH2 groups. The mild hydrolysis employed i n scheme I I (Fig. 3) gives as amide nitrogen only asparagine and other s i m i l a r l y constituted acid amides, i . e . , those having the -GO-KHg groups free. Furthermore in scheme I (Fig* 2) the free amino (both mono- and di-) and also the combined amino nitrogen (both mono- and di-) are determined separatelyi 'whereas i n scheme I I (Fig. 3) compounds containing the combined di-amino nitrogen groups are precipitated with the bases. This l a t t e r method w i l l , therefore, give only the free mono-amino nitrogen* The procedure of Thomas (Figs* 2 and 3) includes also the determination of two not very c l e a r l y defined nitrogenous compounds, v i z * , the so-called "melanin" and "humin" fractions* The term "melanin" i s used to denote the nitrogen belonging to the acid pigment-like bodies found i n the sap of plants. This f r a c t i o n i s precipitated by calcium hydroxide or magnesium - 17 -hydroxide under the conditions defined i n the procedure* The nature of the "huinin" nitrogen w i l l he discussed l a t e r when more detailed consideration i s given to the methods of hydrolysis employed "by various investigators. The procedure of Orcutt and Wilson (Pig. 4), although a much more recent development, does not d i f f e r fundamentally from the methods of Chibnall and Thomas. The chief difference l i e s i n the fact that hydrolysis i s conducted i n two stages. The f i r s t stage of hydrolysis i s accomplished by incubating the material at 40° C. following the addition of a measured amount of enzyme solution, while the second stage i s attained through the aid of sodium bisulphite. The scheme of analysis employed by Mulay (Pig. 5) i s not as comprehensive as those discussed above* No attempt, for instance, is made to determine the exact nature of the compounds contained i n the water soluble material. This method has been devised, with a view to determining the relationship existing between t o t a l soluble, soluble protein, non-protein and insoluble nitrogen within the plant during i t s growing period. Total Nitrogen - The t o t a l nitrogen content of dried plant material i s determined by means of the Kjeldahl method. The chief source of error i n t h i s determination arises through the presence of nitrogen i n the form of nitrates. This error i s usually overcome by f i x i n g the nitrates i n combination with an aromatic compound such as s a l i c y l i c acid. Most widely used of s a l i c y l i c acid modifications i s that proposed by the Assoc-i a t i o n of O f f i c i a l A gricultural Chemists (1925). - 18 -Total Nitrogen of Extracts - Very l i t t l e d i f f i c u l t y i s encountered in the determination of t o t a l soluble nitrogen when nitrates are absent; the standard Kjeldahl method being quite satisfactory i n every respect* The presence of nitrates i n the soluble material, how-ever, gives r i s e to considerable d i f f i c u l t y . Owing to the presence of large amounts of water i n plant extracts, the usual salicylic-thiosulphate method f a i l s to give accurate or consistent results unless certain precautions are taken. In order to obviate this d i f f i c u l t y Banker (1925) recommends the evaporation of an exactly neutral portion of . the extract to dryness i n the Kjeldahl f l a s k prior to making the determination of t o t a l nitrogen* Pucher, Leavenworth and Vickery (1930) i n turn have proposed an adaptation of the reduced iron method for the determination of nitrates i n order to eliminate the inconven-ience involved i n evaporating the extract to dryness. Soluble Protein Nitrogen - The importance to be attach-ed to the determination of soluble protein nitrogen varies considerably i n the opinion of different investigators. Spoehr (1923), for instance does not consider the removal of soluble proteins as being a necessary part of the an a l y t i c a l procedure. Orcutt and Wilson (1936) remove soluble proteins along with chlorophyll and c o l l o i d a l material by heating the expressed sap of the plant to 70° C. Proteins and other colloids s e t t l e out of solution as a coagulum which i s readily f i l t e r e d out upon cooling* The amount of nitrogen removed from the plant tissue by t h i s procedure i s not deter-- 19 -mined. Chibnall (1922), on the other hand, determines the soluble proteins of plant extracts gravimetrically. Proteins are removed by warming the extract to 60° C., cooling, and f i l t e r i n g off the coagulum which i s then washed with alcohol followed by prolonged extraction with ether. The protein i s then dried and weighed* Beagents for the Removal of Soluble Proteins - The reagents most commonly employed as protein precipitants are, copper hydroxide (stutzer's reagent), mercuric chloride, c o l l o i d a l f e r r i c hydroxide, and many acids among which the following are most important, acetic, t r i c h l o r o a c e t i c , phos-photungstic, tannic* p i c r i c , and metaphosphoric acids. The value of any one reagent i s determined by i t s a b i l i t y to effect a complete separation of the proteins with the removal of as l i t t l e as possible of the intermediate products of protein degradation. There i s , however, considerable d i f f e r -ence of opinion as to the most effective reagent for this separation* Thomas (1927) is one of the few workers who maintain that i t i s extremely doubtful i f any one reagent can successfully accomplish the complete exclusion of a l l products, which are only a l i t t l e below the o r i g i n a l proteins i n their complexity. At any rate, the evidence tends to point to the conclusion that a reagent satisfactory for one kind of b i o l -ogical material may be unsuitable for another. Thomas (1927) strongly advocates the use of c o l l o i d a l f e r r i c hydroxide as a means of separating proteins from solu-t i o n . He claims the following advantages for this method:-- 20 -( l ) i t is more convenient and expeditious; (2) i t permits a l l the amino acids to' go through, occluding and precipitating none; (3) i t effects a sharp separation of true protein from i t s decomposition products; and (4) the adsorbed proteins can be recovered quantitatively from the residuum. Hart and Bentley (1915) have shown that Stutzer's re-agent may not, in a l l cases, effect a complete separation of a l l protein and polypeptide structures from amines and amides. There being a tendency for copper salts under certain condi-tions to form copper compounds with certain amino acids. H i l l e r and Van Slyke (1922) made an extensive study of a number of protein precipitants. Working with Witte's peptone, they found that trichloroacetic acid i n solutions more dilute than 5 percent was quite effective as a precipitant for soluble proteins. It was claimed also that trichloroacetic acid per-mitted a l l the intermediary products of protein degradation to pass into solution. Tungstic and p i c r i c acids were observed to be quite effective as protein precipitants but i n addition these reagents had a tendency to remove the intermediate nitrogenous compounds rather completely* Alcohol was found to behave toward Witte's peptone in a fashion similar to tungstic and p i c r i c acids. However, i t was pointed out that such precipitates were inextricably contaminated with other non-precipitable contents' of the solution (e.g., adsorption of free amino acids). Metaphosphoric acid, c o l l o i d a l i r o n , and mercuric chloride were intermediate between trichloroacetic and tungstic acids in the completeness with which they pre-cipitated the intermediate products of protein decomposition. - 21 -Trichloroacetic acid has been used quite successfully as a precipitant of soluble proteins by Wasteneys and Borsook (1924). The concentration of the reagent used was such that i t did not exceed 2 percent of the f i n a l concentration of the solution being analyzed. Proteoses and Simpler Peptides - Plant chemists/as a rule have not attached any particular importance to the pres-ence of proteoses and peptones. They have, almost without exception, considered i t unnecessary to distinguish these fractions from the soluble proteins. It i s recommended by some workers that the proteoses and simpler peptides be precipitated i n common with protein by means of tungstic acid.. In blood analysis, Polin and Wu (1919) preferred this reagent to trichloroacetic acid. Bumsey (1923) used sodium tungstate e f f e c t i v e l y i n the removal of proteins and proteoses from f l o u r extracts when the pH was reduced to 2.0 or less. M e r r i l l (1924) found a somewhat high-er optimal pH was possible when the above method was employed on bacteriological sera. Quantitative removal of proteoses was accomplished by Ghibnall (.1922) following saturation of the protein free plant extract with zinc sulphate. Wasteneys and Borsook (1924), on the other hand, determined proteose nitrogen by difference following saturation with sodium sulphate under r i g i d l y defined conditions. Amide Nitrogen - This fraction i s estimated in terms of the ammonia freed upon the hydrolysis of an aliquot of the plant extract. - 22 -The procedure now generally followed (Tottingham et a l . 1935) j i s to hydrolyze with 10 percent sulphuric acid for 2-§-hours rather than with 6 percent hydrochloric acid as used formerly. This concentration of sulphuric acid furnishes .approximately the same degree of ac i d i t y as 6 percent hydro-chloric acid and possesses i n addition the advantage of not reducing n i t r a t e s . Following hydrolysis the solution i s neutralized with an excess of sodium carbonate and the ammonia d i s t i l l e d over. The amount of amide nitrogen i s found by subtracting from the t o t a l amount of- ammonia obtained, the free ammonia nitrogen determined on a corresponding aliquot. An appreciable source of error encountered in acid hydrolysis i s due to humin formation resulting from the con-densation of tryptophane, ammonia, and other amino acids, with aldehydic compounds such as glucose. It has been observed that humin production i n certain kinds of plant material i s not of s u f f i c i e n t magnitude to introduce an appreciable error. Orcutt and Wilson (1936) found that hydrolysis of soybean juice gave r i s e to 10 to 15 percent humin* In order to reduce the amount of humin formed they undertook a study of various hydrolytic agents such as sodium bisulphite, zinc amalgam, hydriodic acid, and th i o - g l y c o l i c acid. Hydrolysis with. 20 percent sodium bisulphite for 3 hours gave a successful hydrolysis of amide nitrogen with only negligible humin formation. However, they observed that the bisulphite had no hydrolyzing effect whatever on peptide l i n k -ages. This d i f f i c u l t y was overcome by conducting a preliminary - 23 -hydrolysis with the aid of an enzyme solution which was follow-ed "by the "bisulphite hydrolysis* This procedure was found to "be effective i n decomposing peptide linkages completely with the additional advantage of not resulting in humin formation. Amino Nitrogen - i s determined by means of the Van Slyke amino apparatus. Complete descriptions of the apparatus, the technique involved, and the tables necessary for calculat-ing the data are to be found i n the standard texts of Physiol-ogical Chemistry such as that of Hawk and Bergeim. If free ammonia is present i n the solution i n apprec-iable amounts i t must be removed before the determination of amino nitrogen i s made. • Stuart (1935) has reported that certain other sub-stances, among which the polyhydric phenols are important, interfere with the Van Slyke determination by yielding excess-ive "amounts of gas which are measured as nitrogen. Tottingham et a l . (1935) recommend d i s t i l l a t i o n at 45° C. with a s l i g h t excess of calcium oxide with a view to removing or denaturing excess ammonia and polyhydric phenols. Ammonia Nitrogen - The amounts of free ammonia occurr-ing i n plant extracts are usually very small. The deter-mination i s most frequently made to serve as a blank for the amide nitrogen determination. The method developed by Van Slyke (1911) which involves the d i s t i l l a t i o n of the ammonia in vacuo with alcohol and calcium hydroxide i s most frequently used for measuring the amount of nitrogen i n this f r a c t i o n . - 24 -Tottingham et a l . (1935) point out that the chief • source of error arises from the fact that some plants contain appreciable quantities of other v o l a t i l e bases which are carried over with the ammonia i n the usual methods of aeration •or d i s t i l l a t i o n . Nitrate Nitrogen - The methods generally employed for the quantitative estimation of nitrate nitrogen are of three types:-(1) Reduction i n alkaline solution with Devarda's a l l o y and determination as ammonia. (2) Reduction i n acid solution with iron and deter-mination as ammonia. (3) Colorimetric methods. • Various modifications of the Devarda's all o y method are to be found i n the work of Chibnall (1922), Strowd (1920), and Sessions and Shive (1920). Vickery and Pucher (1929) have described a procedure for the determination of nitrate nitrogen i n tobacco by the reduced iron method. The phenol disulphonic acid method i s the most popular of the colorimetric methods for nitrate determinations. Emmert (192.9) has developed a simplified method for use with tomatoe and lettuce while Holtz and Larsen (1929) have proposed a modification for the determination of nitrates i n wheat. It might be said at this point that a method which i s considered to be applicable to extracts of a l l types of plants has been developed by Erear (1930), • Basie Nitrogen - This f r a c t i o n refers to that portion - 25 -of the nitrogen which occurs i n the.form of simpler nitrogen-ous "bases. The exact nature of such oases and the forms i n which they are present are not as yet f u l l y understood. No satisfactory method has been devised, as yet, for studying .them. It i s highly probable that these substances possess considerable physiological significance i n the processes of plant metabolism* The most widely used method for studying basic nitrogen has been by phosphotungstic acid precipitation as o r i g i n a l l y devised by Osborne and Harris (1903). Vickery (1927) undertook a comprehensive study of the nitrogen bases of plants. He found, that with plant extracts, the use of phosphotungstic acid gave a very uncertain measure of true basic nitrogen, whereas when used in mixtures of amino acids obtained from the hydrolysis of proteins, i t i s f a i r l y d e finite in i t s action. C. DISCUSSION AND CONCLUSIONS t-It would appear from the foregoing discussion that, while the general method of approach to the study of nitrogen metabolism i n plants i s the same i n p r a c t i c a l l y a l l investig-ations; the detail s of an a l y t i c a l procedure, as adopted by different workers, vary to a remarkable degree. Consequently the results obtained i n these investigations are such that i t i s exceedingly d i f f i c u l t to draw definite or significant conclusions. The lack of agreement among workers generally with regard to the details of ana l y t i c a l procedure has been due - 26 -very largely to the extreme v a r i a b i l i t y i n composition and physiological makeup of the plant species each has been work-ing with. Because of the unstable nature of physiologically actlvi nitrogen compounds and their complex interrelationships within the plant, attempts to use the methods of pure chemistry i n the i r study give r i s e to considerable d i f f i c u l t y . Therefore, i t i s probably safe to say that a stereotyped method applic-able t.o a l l types of plant material i s impossible of develop-ment. In nitrogen studies particular emphasis has been placed upon the i d e n t i f i c a t i o n of the type of nitrogenous constituents present. The amounts and particular kinds of amino acids present i n the soluble nitrogenous compounds have been deter-mined following the hydrolysis of the material. Comparatively l i t t l e attention has been devoted to a study of the degree to which these amino acids and amides are condensed. In other words, l i t t l e importance has been attached to the det.erminat-' ion of soluble proteins and compounds such as proteoses and peptones which are only s l i g h t l y below "true proteins" i n complexity. Prom,the standpoint of the active metabolism of the plant the degree to which the simple nitrogenous compounds are condensed is probably of equal, or perhaps even greater importance than the free amino acids. It i s reasonable to expect that the determination of "combined" amino and amide nitrogen w i l l not give a t r u l y representative picture of conditions actually existing within the plant at any given time. Protein synthesis or breakdown occurs i n numerous stages giving r i s e to compounds of varying complexity. Consequently the breaking down of soluble nitrogen constituents into the simpler amino acids w i l l not give an accurate picture of protein synthesis and degradation. It may be said, therefore, that the usual methods of studying nitrogen metabolism i n plants do not permit of the determination of the nitrogen compounds as they actually exist within, the plant at the time of sampling. An extract of the soluble nitrogenous constituents of a plant i s , i n a l l pro-b a b i l i t y * representative of an incomplete protein hydrolysate. Consequently methods should be used which permit of studying this material without further modification by hydrolysis. In doing so more attention should be given to the . estimation of the fractions such as proteose, peptone, and sub-peptone which are only s l i g h t l y less complex than the or i g i n a l proteins. A study of this nature conducted throughout the growing and f r u i t i n g periods of the plant would give a cle a r l y defined impression of the rel a t i v e rates of protein degradation and synthesis at different stages i n the plant's l i f e history. This information together with the amounts of the r e l a t i v e l y simple forms of nitrogen existing i n a free state should give a truer picture of the actual nitrogen "setup" within the plant. B. THE .HBTHOS Off WASTMEBYS AM B O R S O O K . In view of the fact that the methods of analysis usually - 28 -employed do not permit of determining the true relationships existing between the various soluble nitrogenous compounds within the plant, the method of Wasteneys and Borsook (1924) is suggested as a possible means of p a r t i a l l y overcoming this - d i f f i c u l t y . Developed primarily for the f r a c t i o n a l analysis of incomplete protein hydrolysates, the method of Wasteneys and Borsook has given consistent and accurate results when used by different workers for a number of years. Proof of the value of the method i s evidenced by i t s ever increasing applic-ation i n the study of the specific changes occurring during cheese ripening (Eagles and Sadler, 1932), (Sherwood and Whitehead, 1934) and (Sherwood, 1935). Wasteneys and Borsook (1924) state that, "the constit-uents of an enzymatic hydrolysate of protein can be divided according to their complexity into s i x fractions! protein, metaprotein, proteose, peptone, sub-peptones, and amino acids". In order to secure more definite information regarding the changes occurring during hydrolysis than i s obtained by the usual free amino determinations, they have devised a method for the quantitative estimation of the above fractions. These workers introduced no new principles but they did so specify the conditions of procedure that quantitative estimation of the above named constituents was possible. The procedure, as o r i g i n a l l y developed, is represented diagrammat-i c a l l y i n Pig. 6. A f u l l description of the method as revised to meet the requirements of the investigation reported herein - 29 -Pig. 6. Procedure devised by Yfasteneys and Borsook for the fr a c t i o n a l analysis of incomplete protein hydrolysates Hydrolysate (1) Total ¥ on 5 cc. samples in t r i p l i c a t e (2) (3) leta'protein pptd. by adjusting reaction to pH 6.0. H". determ. on f i l -t rate. After allowing for d i l u t i o n . H(l)-U(3) = metaprotein. Trichlor. ppt.able N-H(3)= protein 11. Protein pptd. by trichloroacetic acid. N determ. on f i l t r a t e N(l)-U(2) - protein * metaprotein. (4) Proteose pptd. from trichloroacetic f i l t r a t e by ffagSO^'Q 33° C. H. determ. on f i l t r a t e . After allow-ing for d i l u t i o n , H(2) - N(4) = proteose N. (5) Peptones pptd. from f i l t r a t e (4) by tannic acid. B" determ. on f i l t r a t e . N(4) - ¥(5) - peptone He (6) Residual amino acids and simple peptides determ. from f i l t r a t e (5) by alcohol pre-c i p i t a t i o n . 11 in f i l t r a t e = residual T$. ¥ in alcoholic ppte. = sub-peptone 11. - 30 -w i l l "be given l a t e r . B r i e f l y , the procedure involves the precipitation of protein by trichloroacetic acid, of metaprotein by careful adjustment of the reaction, of proteoses by saturation with Ea2S04 at 33° C., of peptones by tannic acid under fixed conditions; and the determination of the residual amino acids and simple peptides by a modification of the alcohol precipi-tation methods of Polin and Denis (1912) and Van Slyke and Meyer (1912)* - 31 -PART I I - EXEERIMENTAL A. OUTLINE OE EIELD EXPERIMEHT t-The a l f a l f a used in this investigation was a strain of .Ontario Yariegated designated as Ottawa, No. 176. The mater-i a l was grown in duplicate rod rows in the forage crop nursery at the Dominion Experimental Earm Agassiz from seed supplied "by the Division of Eorage Plants, Ottawa. The seed was sown in the spring of 1933. Three cutting; of hay were taken.from the rows during the two succeeding seasons of 1934 and 1935 and the 1936 material was used for the study reported herein. Previous to "being l a i d down as a forage crop nursery, the area in which the a l f a l f a was grown had "been i n hoed crop for one year. The s o i l i s a well drained, very lig h t sandy loam. Relatively heavy dressings of well rotted barnyard manure were applied to the nursery f i e l d i n the spring of each year. Stages of Growth at which Samples were taken - Cuttings of the a l f a l f a were made at the following stages of growthr-(1) Seedling stage (2) Pre-bud stage (3) Bud Stage (4) Tenth-bloom (5) E u l l bloom (6) Maturity In order to obtain material for the seedling stage additional seed was procured. This was sown July 8, 1936 i n - 32 -quadruplicate rod rows in order to obtain a quantity s u f f i c i e n t for analysis. The sample was cut six weeks after planting. The plants were approximately six inches high and nodulation of the roots had not taken place. The pre-bud samples were cut at the stage just prior to the formation of flower buds. In order that a l l samples be cut at a comparable stage of- maturity, sampling was carried out at monthly intervals. • Bud stage samples were cut when the flower buds had d e f i n i t e l y formed but none had started to open. Owing to the r e l a t i v e l y long period during which buds are present before blooming actually s t a r t s , t h i s stage i s rather d i f f i c u l t to define* Cuttings were made at as nearly the same stage of development as observation would permit. The tenth-bloom series were cut when approximately one tenth of the flowers were opened out. This stage was compar-able to that at which a l f a l f a hay is usually cut i n normal farm-practice. P u l l bloom samples were taken when no unopened flower buds remained and the ea r l i e s t flowers were just commencing to wither. The mature sample was not cut u n t i l the seed had ripened. By this time the plants were exceedingly coarse and woody in texture. Also considerable leaf dropping had taken place. Total R a i n f a l l Between Cuttings - The t o t a l amount of r a i n f a l l between cuttings at a l l stages of growth are given - 33 -below i n Table I. With respect to the f i r s t cuttings at each stage of growth i t i s to be noted that the r a i n f a l l figure includes a l l the precipitation from the f i r s t of A p r i l , 1936 t i l l the data of sampling. For subsequent cuttings the r a i n -• f a l l i s recorded from the time of the preceding cutting. In the case of the seedling stage the r a i n f a l l i s that which occurred from the date of seeding (July 8) u n t i l the time of sampling. TABLE I . - R a i n f a l l between cuttings. Stage, of Growth _ — - ^ t o ^ . Date of Gutting ;. R a i n f a l l i n inches Seedling Aug. 19 , 1936 1.42 ^ Pre-bud June 6, 1936 July 7, 1936 Aug. 5, 1936 Sept. 9, 1936 10.1G & 4.70 1.28 2.51 Bud June 11, 1936 July 17, 1936 Aug. 19, 1936 11.18 A 4.90 0.14 Tenth-bloom June 13, 1936 July 3'0, 1936 Aug. 27, 1936 11.31 SL 4.77 0.50 P u l l Bloom June 21, 1936 Aug. 5, 1936 13.22 •* 2.86 .Maturity Sept. 9, 1936 18.59 & • From July 8th to August 19, 1936. Total r a i n f a l l from A p r i l 1st to time of cutting. - 3 4 -Method of Sampling - The two-row plots of alfalfa, were divided In such a manner that approximately s i x feet was a l l o t ted to each treatment and each section v/ithin the rows was cut only at the stage of growth denoted by i t s respective la b e l . Consequently samples of any particular stage of maturity taken lat e r i n the season were from the same plants as the e a r l i e r cuttings of that stage.. Immediately after cutting the samples were transferred to special drying trays and dried indoors. . This practice enabled drying to take place at a r e l a t i v e l y rapid rate with-out any appreciable loss in colour or wastage of leaves. The resultant product was a hay of s l i g h t l y better physical quality than alfalfa, hay made under the most ideal f i e l d conditions. 'Following drying a l l samples were packed i n dustproof cardboard cartons and stored i n a dry room. B. ANALYTICAL METHODS t-Preparation'of Material for Analysis - Prior to grind-ing the samples-were subjected to additional drying i n an e l e c t r i c oven at a temperature of 4 5 ° C. for a period of twenty-four hours. This 'procedure--which- reduced the .moisture-content of the material to approximately 3 percent was found to be necessary i n order that a l l the sample might be ground to a uniform degree of fineness. Grinding was carried out with the.aid of a small hand-operated corn m i l l . The grinding surfaces were set as closely as possible and the resulting material was of approximately the same degree.of fineness as .the ground a l f a l f a meal of commerce. Both leaves and stems were ground together and the product thoroughly mixed to insure a uniform sample. These samples were preserved i n t i g h t l y stoppered bottles. Dry Hatter - This determination was carried out i n t r i p l i c a t e . Aliquots of approximately 0.5 gm. were placed i n tared aluminum crucibles, weighed, and dried to constant weight i n a water-jacketed oven at a temperature of 98° C. Total Nitrogen - Quadruplicate one gram samples were analyzed by the Gunning modification of the Kjeldahl method. D i s t i l l a t i o n was made into 0.07143N hydrochloric acid and the excess acid was t i t r a t e d with 0.07143N sodium hydroxide using Methyl Bed as the indicator. Since careful qualitative tests with a c i d i f i e d diphenylamine reagent f a i l e d to reveal any traces of nitrates i n the dried material, the determinations of t o t a l nitrogen were accordingly not modified to include them. - i -Extraction of Soluble Constituents - Duplicate con-secutive extractions were made on each sample, according to the method of Davidson, Clark and Shive (1934). 50 gms. of the f i n e l y ground tissue' were placed i n a 600 cc. beaker, covered with 400 cc. of boil i n g d i s t i l l e d water and heated on a boil i n g water bath for 20 minutes. The content of the beaker were then decanted on to an 18-inch square of cotton towelling suspended over a two-litre beaker and express-ed by hand. The residue was then transferred to a large mortar and the coarser particles ground to a fine pulp with - 36 -the gradual addition of 200 G C . of "boiling water containing 100 cc. of 1 percent phenol. After grinding, the sample was returned to the cloth and again expressed by hand. Following this extraction, the material received a further brief grind-i n g i n the mortar with 200 cc. of b o i l i n g d i s t i l l e d water. It was then-transferred back to the cloth where wringing and washing were continued u n t i l the- t o t a l amount of extract obtained comprised a volume of 1000 cc. The extract was then f i l t e r e d through nitrogen-free f i l t e r pulp i n a Buchner funnel to remove c e l l u l a r material. A clear reddish brown f i l t r a t e free of s o l i d material was thus obtained ( f i l t r a t e 1. Fractionation of Soluble Mtrogen - The procedure used was that of Wasteneys and Borsook as modified by Eagles and Sadler (1932) i n the application of the method to a study of nitrogen d i s t r i b u t i o n during cheese ripening. The modifica-tion as used by them involved a difference i n the size of aliquots and i n the actual amounts of the reagents used for each determination. There i s too, a slight difference i n the conditions under which proteose is determined. Metaprotein was not determined i n the method of Eagles and Sadler, nor was any d i s t i n c t i o n made between sub-peptone and residual amino acids. The scheme of analysis followed i s , for c l a r i t y , represented diagrammatically i n Fig. 7. It is to be noted that this scheme includes the determination of amino and amide nitrogen. The methods used i n their removal are those employ-ed by Eagles and .Sadler (1932). Ho provision, i t w i l l be - 37 -Fig. 7. Scheme of analysis adopted i n this investigation. •Water. Extract of A l f a l f a ( F i l t r a t e 1.) Total >pi.ISP oh 5CG."samples i n quadruplicate Prote ins ppted.by trichloroacetic acid.IT determ.on f i l t r a t e s (3A & 3B) = Total, non-protein 3ST 50cc. port'ion of f i l t r a t e 1.boiled with d i l . acetic acid & f i l t e r e d ( f i l t r a t e 2.) N.determ.on f i l -t r a t e = acetic acid - sol.M Proteose pptd. from trichloroacetic f i l t r a t e s by ¥a2S04 @ 33° C. IT.determ. on f i l t r a t e s (4A & 4B) after allowing for d i l u t i o n F(3A & 3B) -U(4A•& 4B) = Proteose IT Peptones pptd. from f i l t r a t e s (4A & 4B) by tannic acid. H". de.tertm on f i l t r a t e s = sub-peptone N. Kv('4A & 4B) - sub-peptone ET = peptone 1". 50cc. 'of f i l t r a t e 1. treated with 19% P.T.A. & 20% H 2S0 4 f i l t e r e d . N.deterrcuon f i l t r a t e = Amide U. lOOcc. f i l t r a t e 2. treated with 19% P.T.A. & 20% H2S04 f i l t e r e d . . U. determ. on f i l t r a t e = Amino N Total s o l . IS - Total Non-protein - Soluble protein N. - 38 -recal l e d , was made for these determinations i n the original procedure of Wasteneys and Borsook. Total Soluble Nitrogen - Quadruplicate 5 cc. portions of the water extract ( f i l t r a t e l ) were analyzed for t o t a l .nitrogen by the Kjeldahl method. Semi-quantitative tests with a c i d i f i e d diphenylamine reagent f a i l e d to reveal even traces of nitrate nitrogen i n the extracts of any of the samples. It might be added that the nitrate modification of the Kjeldahl method, as proposed by Ranker (1925), was t r i e d but the results obtained were no higher than those from the standard Kjeldahl procedure. Soluble Protein Nitrogen - This fraction was obtained by difference following the removal of proteins with t r i -chloroacetic acid. To 150 cc. of the water extract ( f i l t r a t e l ) were added 30 cc. of 20 percent trichloroacetic acid and the mix-ture allowed to stand for 1 hour. The solution was then f i l t e r e d ( f i l t r a t e 3). The f i l t r a t e was divided into two 70 cc. portions ( f i l t r a t e 3A and f i l t r a t e 3B) which were placed i n a slowly b o i l i n g water bath for 3 hours to decompose the trichloroacetic acid, and to drive off the resulting carbon dioxide and chloroform. Bach f l a s k was then cooled to room temperature, f i l t e r e d and ms-de up to the o r i g i n a l 70 cc» Volume. Two 5 cc. portions of each f i l t r a t e , f i l t r a t e 3A and 3B, were taken for the nitrogen determination ( t o t a l non-protein nitrogen). In this way results in duplicate of duplicate portions were obtained. Soluble protein nitrogen (trichloroacetic acid pre-- 39 -cipitate) was calculated by subtracting the t o t a l non-protein nitrogen from the t o t a l soluble nitrogen after making correct-ions for d i l u t i o n . Proteose Nitrogen - To the remainder of f i l t r a t e s 3A - and 3B respectively (approximately 60cc.) 22 gm. of anhydrous sodium sulphate were added. The mixtures of salt and solution were held at 35° C. for 1 hour and then f i l t e r e d into 25 cc. volumetric flasks through a water-jacketed f i l t e r maintained at 33° C. ( f i l t r a t e s 4A and 4B). These were at once washed quantitatively into 50 cc. volumetric flasks and made up to the mark. Two 10 cc. portions of each f i l t r a t e , f i l t r a t e 4A and f i l t r a t e 4B, were taken for nitrogen determination. The difference "between these determinations, after allowing for d i l u t i o n and volume change due to heating, and the nitrogen results of f i l t r a t e s 3A and 3B respectively i s the proteose nitrogen. In calculating the results of the proteose nitrogen the volume increase due to the heating of the solution to 33° C. must not be overlooked. Correction for this volume .change i s made by comparing the volume of water at 20° C. with that at 33° C. when saturated with sodium sulphate. Eagles and Sadler (1932) found that 22.5 cc. of. water at 20° C. when saturated with sodium sulphate at 33° C , occupied a volume of 25 cc. Therefore, the factor for volume increase due to heating i s 25/22.5, Peptone and Sub-peptone Nitrogen - 25 cc. of each of the f i l t r a t e s 4A and 4B were pipetted into 250 cc. Erlenmeyer f l a s k s , to each were added 25 cc* of 2.21 normal sodium hydroxide, and 125 cc. of 20 percent tannic acid dissolved i n o.l normal sulphuric acid containing 20 percent sodium sulphate The mixtures were thoroughly shaken and allowed to stand at -20° G.' for 3 hours and then f i l t e r e d . Two 50 cc. portions of each of these tannic f i l t r a t e s were taken for nitrogen deter-mination. The nitrogen i n the tannic acid f i l t r a t e i s sub-peptone  nitrogen . The difference between the sub-peptone determinations and the t o t a l nitrogen determinations on f i l t r a t e s 4A and 4B respectively i s peptone nitrogen (tannic acid precipitate). Acetic Acid-Soluble Nitrogen - To 50 cc* of the water extract ( f i l t r a t e l ) were added 15 cc. of 1 percent acetic acid and 100 cc. of water. The solution was heated i n a b o i l -ing water bath for 30 minutes, cooled, made up to a volume of 250 cc. and f i l t e r e d ( f i l t r a t e 2). Two 50 cc. portions of this f i l t r a t e were taken for the determination of acetic acid-soluble  nitrogen. Amino Nitrogen - To 100 cc* of f i l t r a t e 2 (acetic acid f i l t r a t e ) were added 30 cc* of 25 percent sulphuric acid (one volume of concentrated sulphuric acid, four volumes water), 10 cc. water, and 10 cc. of 19 percent phosphotungstic acid. After standing 24 hours the precipitate was f i l t e r e d , and two 50 cc. portions of the f i l t r a t e were taken for the determinat-ion of amino nitrogen (phosphotungstic f i l t r a t e ) . Amide Nitrogen — To 50 cc. of the o r i g i n a l water extract ( f i l t r a t e l ) , 30 cc. of 25 percent sulphuric acid, 10 cc. of water, and lOcc. of 19 percent phosphotungstic acid were added. After standing 24 hours the solution was f i l t e r e d . Four 10 cc. aliquots of the f i l t r a t e were taken for.the : estimation of amide nitrogen (phosphotungstic f i l t r a t e ) by the 'Kjeldahl method. Metaprotein Nitrogen - In the o r i g i n a l procedure of Wasteneys and Borsook (1924) this f r a c t i o n was precipitated by adjusting an aliquot of the o r i g i n a l solution to pH 6.0 the point of greatest i n s o l u b i l i t y of albumen acid metaprotein. Owing to the fact that the pH of the a l f a l f a extracts was, on the average, around pH 6.0 metaprotein could not be determined i n this way. Several attempts were made to remove the metaproteins with an i s o - e l e c t r i c point of pH 5.4 (Cole, 1933) by adjusting the reaction of the extract to this pH. No results were obtained however. o. B X E E R H E E N T A L R E S U L T S i -The nitrogen fractionation results are presented i n tables I I to VII incl u s i v e . In table I I the results obtained from the application of the method of Wasteneys and Borsook to the study of nitrogen d i s t r i b u t i o n i n a l f a l f a are expressed i n percent of the absolute dry weight of the material. Table I I I shows the d i s t r i b u t i o n of acetic acid soluble nitrogen, amino nitrogen, and amide nitrogen i n percent, of absolute dry weight. Tables IV and V give the corresponding results of tables I I and I I I i n percent of the t o t a l nitrogen of each respective sample at the stages of growth defined*. Tables VI - 42 -Maturity-r x j UJ C M (-• O H* o B 1/10 Bloom t r i c p . H j H CD I o J £ P. Seedling, Stage of Growth j Sept. 9 ,1936 £ £ • 0Q P i 0 CD • o i £ 2 '(-> h-> to to OJ O l 0 1 O i .!» H c_i .£ £ £ 0 9 (-> p i »• CD \» V» , 1M (-" 0-> t o t o t o OJ OJ O l O i o> »> <H <H £ £ £ H p5 j » «<i CD tO ^ 3 P S \» v> H H H t o t o t o O J OJ OJ O i O i O i June 6,1936 July 7,1936 Aug. 5,1936 Sept.9,1936 > . « r — ' t o *» \-> t o OJ O i c+ <r+ c+ CD H-C i o CK3 Hi . to to to ' w o • o ' - 3 •>}• ro to • » « o> to O i <j o O i OJ ro to . « «. < J CO < J !to O i <j O) 0-" .OJ OJ ro . » © O i CO O ! 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Ol tt ' 9 ' ' 9 t o t o O l O O l t o i f* t o OJ t o • e e © 0 o i o to 01 tO -d Oi H H H O l t o t o 9 9 0 H OJ H tO O J t o OJ t o i f * OJ 0 0 0 0 d2" O l i t * - 3 H O H i t * t o t o t o t o O l O OJ to 9 9 9 9 O tO <2 tO O J -<J O H OJ t o O i OJ o OJ O i t o Ol o Oi Oi CO Q cr hf p> O TO =3 CD cr P * o O t ) P P cr cr cr C D H» O o — ' H CO : o ^ H P P <n-c y C D H h i C D H3 o C T p H H J p h i to o o "fej c+ H C D p H o * P H C D hj Co h i O O H tei c+ P C D a1 H ' H P C D hj H h i O O c+ «+ P C D H H -P fej o P i h i o CD O to CD Hi j CD •d' c+ O P CD CD p e+ |-o p CD h3 H < ! H» c+ ti o TO CD P u H -to d -. h i c y p c+-(-'• o p H P P H . Hj P H H j P M P •<! o & P w H< H j H j CD h i CD P c+ CO ci-P TO CD CD O H j O 13-- 45 -CO w P CD 0 •ZS 6' I-1 f-> CD rt CQ P £+ P 4 c i - CD ts' CD CD CO e+ 4 P CD 0*3 CO CD to CD O P< H> H-0*5 4 o ' d si CD c+ 4 O CD 0 CD c+ 4 P CD 0*5 CD P-CD G H> Hj H» P" ct-CD S3* P- CD 0 o c+ P P H-c+ 4 o 0*3 CD 53 O Hj CD O t J * 4 CD CO »p CD O e+ CD p-4 C + CD to to tO Oi CO r o ro W w • -03 CO W i t * , o W CO « CO r o o oi co i — 1 bd H o o B P 0*3 I CD CO O l t-> to to W O J o> Oi co r o * * oi CO Oi - 3 O Oi O) OJ CO o « 9 O J M O i CO OJ ro G CO « 9 Oi Oi O l to CO CO tO CO • • 9. 03 I—* O J CO H» o bd o o B > <4 <H £ P P « H p • t<j (j, CO OJ l — 1 X J O OJ M> V «• f-» »-> tO t o t o O J W O ) Oi Oi Oi bd p p , 4 CD c p p« £ p oq i—' 9 «<< M H-' tO - 3 2 CD f-> l _ i | _ i to t o t o W W W O i O O i w ro ro 9 9 « ->3 CO ro o> - 3 Oi j-> <j W W O l 00 O l d> '9' 9 9. G i£» -<2 rfVCJl ; w w ro 9.. 9 9 Oi CO w Ol |J> -<2 O l ii> O l to i> c_i CD p p W 0*5 H* C + 9 « < CD t O O l - J O i f~' H 1 I—1 f—1 tO tO tO tO W W W O J Oi Oi Oi Oi CO CD CD P. H-D 0*5 P 0*3 f-» tO f-> to w Oi w w iJ> r o I—1 co w cb O l I—1 W CO CO O l W O i u u * . O O i 9 9 9 O CO O i t o t o w t!> W rf^ |J> O H d> • *J V*. *> to W CO CO <J CD H © O l w o h- 1 9 CO f—' w w o i O l O o " : 9 « CO ii> i t * O i iJ> - o CO W il> w w w w co o i •v2 CO O W O ) * • O i H O a • 9 O l CO -<! W tO O l CO I—1 W CO e 9 • i»> CO co Oi r j i CO W , Oi O <J • • • t o ^ w ^ CO i - 1 ro (t* tO if> o • 9 9 W tO <2 1—1 Oi O l w w w w W O CO <2 9 * • 9-00 O l Oi 0 1 Oi ^ M i — 1 ro co <J |i> to 9 9 « CO - 3 H 1 O |J> o W CO w w o W O l CO « e e « W it* CO O i O M <3 |J> w O l 9 t o t o w I-1 t o co ro w w Ol W OJ H o e e © -»3 W CO Oi w <J Co co w o -•3 t o CO Q e+ 4 P O 0*3 S! CD ef-ts' o H) • o G p c+ CD erf-H * O 0 H j OQ t s | i-3 o - c+ * ^ p p p^ I—1 CD CD 4 ! s j CO o (-> I H3 O CO o CD tej o P-o CD c+ H ' O 4 hd B P c+ f-3 CD • ^ > tej H i P t-3 CD t> is! H-B P. CD P -!> t-1 P H> P u H» CQ c+ 4 H -P1 c+ H-O P5 - 4 6 -hi CD CD CQ , CQ »C$ P CD !—1 O c+ c+ CO < p CD hi CD CQ am CD am X •b H hi CD CD CQ P CQ c+ CD P* c+ H CD pi CQ *S e+ CD P hj OQ O CD CD P": O e+ Hj P OQ era CD hj O O Si H> r i -ps' c+ P$* CD CD hi c+ CD O e+ H P CD H Hj H< CQ 13 O CD H P. P \ cr H (D 0 H* e+ hi O CR! CD P O Hj CD O Maturity-Pull Bloom 1/10 Bloom Bud Pre ••'bud Seedling Stage of Growth. Sept. 9,1936 June 21,1936 Aug. 5,1936 June 13,1936 July 30,1936 Aug. 27,1936 June 11,1936 !• July 17,1936 Aug. 19,1936 June 6 , ' 1936 July 7, 1936 Aug. 5, 1936 . Sept..9, 1936 ["  11 .."———————:—J © H to ** H to OJ Oi Date of Cutting CO i-3 0.774 0.686 0.861 1.463 1.014 1.416 1.060 1.418 1.103 O-i (-J (_• K_I « • • « to to to to to o ro <3 tO H OJ O l 1.867. otal Water oluble ' W h3 87.80 co <? i t * Ol 9 • ft o t o en to t o oo © • . e Ol to t o <3 O tO oi oo o> o i to-a © © • -•3 tQ 1—1 CO t o o 1 ~3 CO -0 CO to to o to © © «- © OJ CO to CO *OJ H Oi OJ 78.35 otal non-rotein IT H H » O —3 H ro Ol i f* o » t o o i t * CO 19.08 8.80 23,43 OJ H OJ i t * -3 ro • • • 0 to o to M HO tO hr> ?0 i-> O "-3 to -O © © © © -«3 H i t * -~3 21.65 Soluble Protein IT to o » ro GO 12.19 20.41 I-1 H i t * H t o . • , e -3 tD Oi tO tO -3 H CO tO H e « © tO O -3 tO OJ H to co cn Oi • © • • to ro to Oi H OJ ro to -3 © to ro Proteose IT 15.81 H H H Ol a 9 G> 03 tO i f* H H H i t * ro co . . . HOOI O H O 12 a 38 21.55 6.30 CO ~3 to CO • e ©• e OJ to to i f* O) i t * Ol -3 8.16 hd CD e+ O CD 52.85 49.89 51,96 '• 52.75 67.20 47.68 Ol Ol i f * o ro o i O 9 0 i t * i t * o tO H H • Oi Oi Ol Oi H Oi OJ -3 © • • , H Oi OJ O) O i t * tO ~3 Oi ro e ro -3 CD p e h 1 O 0 CD H I fe. H c+ hi O 03 CD 0 u H-CQ tH-hi H P c+ H O P -H-P5 > H Hj P H Hj p P «< G c+ P c+ w H-Hj H , CD hi CD Pi c+ CQ c+ P oq CD CO o Hj Q i-i o si ci -ts' - 47 -to 1 ' P H CO p c r c+ P T CD W CD to P H «rr to P hi CD P c r P hi H ' c r CO CD £+ «3 H t o OJ CD <2 it* tO CO 9 - 3 tO tO OJ - 3 t o OJ tO H H bd H O O 3 > o b d H O O g P P TO p • CD t o Ol H H J - 1 tO tO OJ OJ Oi Oi 0 o e - • CO Oi 01 CO H OY t o t o it* O l « • - a t o o o i t o - 3 to OJ 9 0 H (t* CO (-» -vj Oi t o o ' • - * • O l Oi o> t o P P TO H P P CD bd P ft tO OJ j-> -«3 O O) J_l | _ 1 tO tO tO OJ OJ OJ Ol Oi Oi H H H • » • it* O it* H H O l oi it> OJ P P P TO H a • >-> M H tO »0 I H *o »*> H H H tO tO tO OJ OJ OJ Oi Oi Oi hi hi CD I cr p ft H H H 9 » • -H it* O O H O ) O J CO o 'CO >• C-) CD p p p to' TO • H» P c+ « ^ CD tO Ol - 3 Oi t-> h-' h-1 tO tO tO tO OJ OJ OJ OJ Oi Oi Oi Oi to CD CD ft H H -P TO P H tO , H tO OJ OS t o o t o O O O i 9 0 0 H O CO w o o OV CO <2 tO tO - 3 0 9 0 co t o O J 01 <£> - 3 Oi - 3 O l - a Oi -a 0 - 0 0 CO O O l it* O O l -<2 tO tO - o o - o 9 - * Oi t o it* Oi M CO H H I—' H 9 9 0 9 ' t o t o t o t o tO O tO -C! tO H O J O l CO Oi - 3 co t o co co tO <i Oi O l 9 0 9 0 Oi H Oi O ft? H H -<3 CO <! OJ it* Oi Ol tO it* < S H 0 0 0 H Oi O J CO Oi t o Ol Oi Oi t o -<J Ol 0 0 « H O tO CO - 3 H - 3 <3 - 3 - 3 O J it* tO O J 0 9 - 9 e t o O J it* co 01 CO C l it* ~3 - 3 « H it* o i -JI ~a ~3 tO H O i H 0 0 9 o CO O O J O i H - 3 if* -<3 it* e ->3 H CO Q c h h j P o TO ti CD c r t ? O H) o U p p c r c+ c r CD H> P O TO H J 02 H3 O O H c r P P c r H H .. CD P tej c+ CD r~^ hs t o o O CD H c r P H * c r o H CD {> o ft 0 » > 1-3 g t> p 0 o H> fej H H c+ hi p c r CD •hi: ' 0 p> c+ H3 g h! 0 H -P [b- P i c r o CD CD H> - — H * te{ H 1H-3 6 y <! H H P H CO i> c r H hi H J H -P cr H p H > c r P H -o p o H ) O CD c r H O W > H > O Hj H -CD hi CD P Cr W P o >p c r P ft CO o H P CT CD to c r P 09 CD «tej tfl H -e+ O hi H j O TO Q CD hi 0 O w Si {3* g P O H> C + hi o TO CD P P P ft 6* H> ft CD fel H » c+ hi o TO CD P - 48 -and VII contain the results of the water soluble material expressed i n percent of the t o t a l soluble nitrogen of each respective sample. In connection with these tables the following d e f i n i t -i o n s must be noteds-(1) "Insoluble Protein nitrogen" i s calculated by-subtracting the t o t a l water soluble nitrogen from the t o t a l nitrogen value for each sample. (2) ^.Soluble Protein Nitrogen". This fraction i s obtained by subtracting the nitrogen of the trichloroacetic acid f i l t r a t e from t o t a l water soluble nitrogen. (3) "Total Non-Protein Nitrogen" i s the t o t a l nitrogen of the trichloroacetic acid f i l t r a t e . (4) "Proteose Nitrogen" i s obtained by difference follow-ing saturation with sodium sulphate under r i g i d l y controlled conditions. The nitrogen of the sodium sulphate f i l t r a t e i s subtracted from the t o t a l non-protein nitrogen (trichloroacetic acid f i l t r a t e ) . (5) "Peptone Nitrogen". The nitrogen'precipitated by tannic acid under the conditions of the experiment. It is determined by subtracting the nitrogen of the tannic acid f i l t r a t e from the nitrogen of the sodium sulphate f i l t r a t e . (6) "Sub-peptone Nitrogen" . This fraction is represent-ed by the t o t a l nitrogen i n the tannic acid f i l t r a t e . It includes peptides of lesser complexity than the peptones i n addition to free amino acids. (7) "Acetic Acid Soluble Nitrogen" represents the - 49 -nitrogen content of the water extract of a l f a l f a after i t has been boiled with very dilute acetic acid. (8) "Amino Nitrogen". This has been defined as that nitrogen unprecipitated by phosphotungstic acid from the •acetic acid f i l t r a t e . (9) "Amide Nitrogen" i s that nitrogen not precipitated by phosphotungstic from the o r i g i n a l water extract. P. DlSGUSSIOIf AND CONCLUSIONS.* Total Nitrogen - An examination of the t o t a l nitrogen data of Table I I when portrayed i n graph form (see Pig. 8) shows a r e l a t i v e l y steady decrease throughout the growing period. A detailed study of the graphs for the three cuttings indicates that, i n the case of the f i r s t cutting, a marked decrease i n nitrogen occurs during the early period of growth followed by a gradual decrease to maturity, except in the case of the tenth-bloom where a sli g h t increase i s noted. The chart for the second cutting, on the other hand, indicates a relative-l y uniform decrease i n nitrogen content throughout the period of study, while the graph covering the th i r d cutting demonstrate very l i t t l e change i n nitrogen content throughout. The fluctuations i n t o t a l nitrogen, which are apparent in Pig. 8, may be p a r t i a l l y explained i n the l i g h t of the work of Woodman and Evans (1935), who found that the proportion of leaf i n relati o n to stem f a l l s off as the crop advances i n maturity. Since the t o t a l nitrogen content of the a l f a l f a leaf at any stage of growth i s , roughly, double that of the F T 2r T O T A L N l T R O C t E h OF A L F A t F A HAY C U T A T 11:J1111 ^^^k .REJT ' lT •STAO.t iS Q F O f i O W T J c i j - 51 -stem, a change i n the ra t i o of these two i s very significant,, Woodman and Evans observed the rate of decrease of leafiness to be greater before budding than during the period of budding to early flower. This accounts for the sharp decrease in t o t a l nitrogen to the tenth-bloom stage i n the second cuttings and the decrease from the seedling stage to budding in the f i r s t cuttings. From budding onward t o t a l nitrogen does not decrease as sharply as before and the decrease can, perhaps, be partly accounted for i n l e a f - f a l l . The r e l a t i v e l y rapid increase i n t o t a l nitrogen between budding and early flower (tenth-bloom) in the f i r s t cuttings indicates that there has been an increased nitrogen intake by the plants at this time to take care of the needs of flowering. Furthermore, since only two days have elapsed between the taking of these samples there was in s u f f i c i e n t time for an appreciable change in leaf-stem ratio to occur. In the second cuttings a futher decrease i n t o t a l nitrogen takes place between budding and early flowering. However, i n view of the fact that the actual amounts of t o t a l nitrogen are much higher i n the preceding stages of growth than i n the case of the f i r s t cuttings there is evidence that the plants already contained a su f f i c i e n t supply of nitrogen to enable flowering to take place without further intake. Also, the extremely slight change i n tota l nitrogen between bud formation and the commencement of blooming i n the third cuttings might be explained on this basis. The data presented by Woodman and Evans (1935) can also be used i n interpreting the increase i n t o t a l nitrogen content - 52 -of the plants at each stage of growth with cuttings made later in the season. They attributed this r i s e to the fact that later cuts i n the season have a d i s t i n c t l y l e a f i e r character than the early cuts. The high nitrogen content of the third cuttings together with the negligible fluctuation between different stages of growth indicates not only a high degree of leafiness i n the plants but, i n addition, that the ratio of leaf to stem does not change appreciably at this time. Observations i n the f i e l d at the time of sampling showed that the later cuts did not achieve the same degree of vegetative growth as the e a r l i e r cuts. From the t o t a l nitrogen data presented i n Fig. 8 certain conclusions can be drawn with respect to the quality of a l f a l f a hay cut at different stages of growth and at different times i n the season. The high nitrogen content at the seedling stage i s of no p r a c t i c a l value since the crop could not be u t i l i z e d for hay at this stage. This i s also p a r t i a l l y true of the cutting of hay at the pre-budding stage. Hay cut a i this stage would have a very high digestible crude protein content, but the y i e l d of dry matter would be very low and also continued cutting at this stage of growth would tend to deplete the v i t a l i t y of the stand rather seriously (Woodman, Evans, and Norman, 1933). In the early part of the season i t i s evident that, from the standpoint of t o t a l crude protein, the cutting of hay i n the one-tenth bloom stage would give the best results. Later in the season i t would probably be permissible to cut at the bud stage in preference to the terath-bloom, pa r t i c u l a r l y under conditions similar to those at the - 53 time the second cuttings were taken. The effect of cutting at an e a r l i e r stage of growth la t e r i n the season would probably not be so harmful to the vigour of the stand as i t would be e a r l i e r i n the season. Later cutting i n the early part of the 'season would permit proper crown and root development to take place. I f possible, hay should not be cut i n the f u l l bloom stage i f a high content of digestible crude protein is to be obtained. Total Soluble Nitrogen - The results for t o t a l soluble nitrogen (see Table IV") are expressed graphically in Figure 9. In order to emphasize the fluctuations between each stage of growth the results are plotted i n percent of the t o t a l nitrogen of each respective sample. It i s of extreme interest to note that the respective levels of t o t a l soluble nitrogen between cuttings, at corres-ponding stages of growth, are the exact reverse of the order existing for t o t a l nitrogen (Pig. 8). The proportionate amounts of t o t a l soluble nitrogen, at nearly a l l stages of growth, are higher i n the f i r s t cuttings than i n the second and thi r d cuttings. The t o t a l soluble nitrogen at the pre-bud and f u l l bloom stages does not follow the general trend for the other stages of growth. Prior to bud development the proportionate amounts of t o t a l soluble nitrogen are p r a c t i c a l l y identical i n the f i r s t and second cuttings. The second cutting-at the time of f u l l bloom appears to possess a s l i g h t l y higher percentage of i t s nitrogen i n a soluble form than the f i r s t cutting. T O T A L y ) l 1 F H I T P O Q E t l O E L A L E A L E A J j A Y t j C U T A T P I F F F R F N T S W . P . s r>F r p r y T H - 55 -Attention must also be drawn to the fact that the proportion of t o t a l soluble nitrogen i s much higher at tenth-bloom than budding i n a l l three cuttings and i n the f i r s t and t h i r d cuttings soluble nitrogen attains i t s maximum value at 'this time. This r i s e undoubtedly accounts, in part at least, for the increase in t o t a l nitrogen (Pig. 8) at the correspond-ing stage of growth i n the f i r s t and t h i r d cuttings and thus further strengthens the assumption, made previously, that there is ah increased nitrogen intake by the plant during early flowering. It has been mentioned above that t o t a l soluble nitrogen increases from budding to early flowering i n the case of the second cuttings whereas t o t a l nitrogen actually decreases. Prom th i s i t may be concluded that a certain amount of protein degradation takes place in order to create a readily trans-located supply of simpler nitrogenous compounds to f u l f i l l the requirements of flowering when the plants are unable to meet this need by increased nitrogen intake. The marked decrease in the proportionate amounts of t o t a l soluble nitrogen from early to late flowering indicates that the excess of soluble nitrogenous material has been u t i l -. ized i n normal plant processes such as; preparation for seed setting, and increases i n f i b r e and stem growth; The s l i g h t r i s e i n soluble nitrogen at the time of maturity might be indicative of protein degradation i n order to translocate nitrogen to the seeds already formed. On the other hand i t may be caused by general breakdown following the cessation of a l l growth processes. - 56 -.With regard to general metabolism the curves for t o t a l soluble nitrogen tend to show an increasing rate of synthesis and assimilation up to the period of early flowering. By this time the- plants have p r a c t i c a l l y achieved their maximum veget-a t i v e growth. Prom this point onwards a consolidation of soluble materials takes place and active growth processes gradually slow down; The probable effects of soluble nitrogen on hay quality are not easy to define. The high proportion of soluble nitrog-en when the plants have just started to bloom indicates that large leaching losses might be incurred in making a l f a l f a hay at this stage of growth under f i e l d conditions. As for the influence of soluble nitrogen on the feeding value of a l f a l f a hay no deductions can be made without further information concerning the exact nature of the compounds con-tained in this f r a c t i o n . However, i t i s generally conceded that the simpler nitrogenous compounds possess a lower n u t r i t -ional value than the "complete"* proteins. Insoluble Protein Nitrogen - Subtraction of t o t a l soluble nitrogen from t o t a l nitrogen (see Tables I I and IV) gives what might be termed Insoluble Protein Nitrogen. Since i t i s determined by difference, the curves for this fraction (Pig. 10) w i l l obviously show trends that are intermediate between those for t o t a l nitrogen (Pig. 8) and t o t a l soluble nitrogen (Pig. 9). It i s apparent that the proportionate amounts of insoluble protein are greater at a l l stages of growth in cuts made later i n the season. This explains the inverse r e l a t i o n -( 0 - I N S O L U R I F P R O T F I N N l T B n r ^ u ^ p f f l p Q - 58 -ship reported previously with respect to t o t a l soluble nitrogen. In general, insoluble protein shows a decrease through-out the periods of active vegetative growth and di f f e r e n t i a t i o n . Following the inception of flowering i t rises markedly thus Validating the assumptions made i n the foregoing discussion with respect to soluble nitrogen. A high proportion of insoluble nitrogen i s not necessar-i l y correlated with hay quality. Hays cut at f u l l bloom or later would have a high content of insoluble protein and, according to Nelson (192.5), cutting at these stages would produce a higher y i e l d of protein per acre than would be obtain-able from cuts made e a r l i e r . However, Woodman, Evans and Norman (1933) point out that, owing to the exceedingly high proportion of f i b r e at these stages of growth, the feeding value i s extremely low. The results obtained i n t h i s investigation show that cuttings at r e l a t i v e l y early stages of growth made later i n the season contain a high content of insoluble protein and in view of the findings of Woodman and Evans (.1935), who have shown that such cuts contain a very low proportion of f i b r e , the feeding value w i l l , i n a l l p robability, be very high. Distri b u t i o n of the Soluble Nitrogen Fractions of  Wasteneys and Borsook i n Terms of Total Soluble Nitrogen - Fig. 11 shows the proportionate d i s t r i b u t i o n of the various soluble nitrogenous constituents i n the f i r s t cuttings of a l f a l f a , as expressed i n Table VI, at various stages of growth, while Fig. 12 depicts the di s t r i b u t i o n of these same fractions at the stages of growth from which second and th i r d cuts were taken. IG I I - D I S T R I B U T I O N OF T O T A L NON-PRQTFIN <Sf)H/fi[ F PROTEIN, P R O T r W , P F P T O J I L ^ ? . 5U8-PEPT0MF miTgnpfjl /Total Pion-profein f t Fiail-PISTRlBUTIONnFTOTAl ^ f^f lT^ l J l^mUDLL ffi^l^PRQTTiQ^PFFTO^^ ...trogeti - It i s evident, that at a l l stages of growth, soluble protein represents a r e l a t i v e l y small proportion of the t o t a l soluble nitrogen. In the f i r s t cuttings a decline i s evidenced between the seedling and pre-budding .stages of growth whereas during the subsequent intervening period, that i s up to bud formation, soluble protein rises very sharply to a peak represented by 3 3 percent of the t o t a l soluble nitrogen. 'At tenth-bloom soluble protein drops sharply to a lev e l only s l i g h t l y above that existing i n the pre-bud stage. During the period of flowering the rate of soluble protein synthesis apparently accelerated and an appreciable increase is to be noted by the time the plants have reached the period of f u l l bloom. From f u l l bloom to maturity a decrease i s indicated thus providing evidence of a certain amount of protein hydrolysis i n view of the fact that t o t a l soluble nitrogen increases at this time. In the second cuttings a sharp decrease from pre-budding to early flowering takes place. This trend is somewhat the reverse of that reported for the f i r s t cuttings, nevertheless i t i s to be expected in view of the observations that have been made concerning the fractions previously discussed. The con-ditions prevailing between tenth-bloom and f u l l bloom at the time the second cuts were taken are apparently comparable to those of the f i r s t cuttings. From Fig. 12 i t i s to be seen that the trend of soluble protein i n the third cuttings i s the same as that encountered in the f i r s t cuttings which are made much ea r l i e r i n the season. At this point attention should also be drawn to the fact that ~ 62 -the proportion of soluble protein at this time d i f f e r s only s l i g h t l y from that existing i n the f i r s t cuts. The high level of soluble protein encountered at the bud stage i s rather significant since this period in the plant's - l i f e h i s t o r y , as already noted, i s generally considered as representing the peak of vegetative growth. Prior to this time the energies of the plant have been devoted c h i e f l y to the production of leaves, stems and roots. Following the appearance of flower buds, however, the plant tends to expend the greater part of i t s efforts i n the formation of f r u i t . Since this phase of development constitutes a drain on the vegetative resources i t i s natural that soluble proteins should be dimin-ished in amount. In so far as quality of hay i s concerned i t i s apparent that the high proportion of soluble protein nitrogen found at the time of budding i s coupled with a low content of soluble nitrogen and a reasonably high proportion of insoluble protein. On the basis of previous discussion the value of such a combin-ation i s obvious. Total Non-Protein Nitrogen - This fraction does not warrant detailed consideration since i t includes almost a l l of the soluble nitrogenous material and, for this reason, i s obviously complementary to soluble protein nitrogen. Proteose Nitrogen - In view of the fact that proteoses are only s l i g h t l y below proteins i n complexity, i t might be expected that the trends for the two would have a tendency to approximate one another. However, this relationship does not hold, except i n two instances. In the f i r s t cuttings proteose - 6 3 -follows a trend similar to that exhibited by soluble proteins up to the time of f u l l bloom. Following the period of flower-ing the situation i s reversed and the quantity of proteose at maturity i s almost double that of soluble protein. An inverse , relationship .exists from pre-budding to tenth-bloom i n the second cuts and from then to f u l l bloom the relationship i s f a i r l y constant excepting that the order of magnitude of the two fractions i s altered. In the th i r d cuttings the trends of soluble protein and proteose follow a course similar to that prevailing i n the second cuttings. It i s worthy of note that the type of graph for proteose i n a l l cuttings i s essentially the same and indicates a d i s t -inct increase throughout the growing period? which i n turn suggests that this f r a c t i o n i s present as a result of both protein synthesis and degradation. The increase throughout the period of active vegetative growth indicating that proteoses may represent one of the steps i n protein synthesis; whereas the continued increase following flowering i s indicative of protein degradation. However, this conclusion tends to be contradictory to the findings of Ghibnall (1922) who observed that proteose in the leaves of the common bean (Phaseolis vulgaris) tended to decrease with increasing age in common with the leaf proteins. Nevertheless, i t i s possible that the mature plants analyzed i n this investigation had not reached the same stage of degeneration exhibited by those of Ghibnall and consequently the increase in proteose may be taken as being indicative of the commencement of degeneration. - 64 -Peptone Nitrogen - In the f i r s t cuttings the curves for proteose and peptone nitrogen are of pra c t i c a l l y the same order throughout the growing period. However, at tenth-bloom there appears to be an appreciable difference i n favour of -higher peptone content, while at maturity, the graphs would appear to suggest s l i g h t l y less peptone nitrogen. With respect to the thir d cuttings i t w i l l be observed that the graph i s of approximately the same order as that of the f i r s t cuttings but, i n this case, the peptone nitrogen content appears to be s l i g h t l y lower than that of proteose nitrogen. The graph covering the second cuttings does not conform to the other curves i n that i t appears to demonstrate a very definite negative correlation between proteose and peptone at the stages of growth from which comparisons may be made. In view of the fact that peptone exhibits considerable i r r e g u l a r i t y throughout the period in which samples were taken i t i s rather d i f f i c u l t to draw any definite conclusions as to i t s probable significance i n nitrogen metabolism. Furthermore the writer has been unable to f i n d any record i n the lite r a t u r e of such a determination having been made on plant material. Consequently no interpretation may be made i n the l i g h t of previous work. However, the di v e r s i t y in the results obtained may be p a r t i a l l y explained when i t i s remembered that the material analyzed had been subjected to a prolonged drying at ordinary temperatures thus permitting enzyme action to proceed unchecked for a considerable length of time. Since, i n a l l probability, - 65 -the amounts and combinations of enzymes present w i l l vary considerably depending on the stage of growth and the time of season at which the samples are taken, i t i s reasonable to expect that the results of their action w i l l be by no means • constant, Neverthelessj peptone, in common with proteose, does show indications of being rather closely correlated with protein synthesis and degradation i n a l l cuttings© Therefore, i t i s quite possible that determinations on fresh plant material would show this relationship more d i s t i n c t l y . Sub-peptone Nitrogen - It i s quite apparent from an examination of Tigs. 11 and 12 that sub-peptone constitutes a very large portion of the t o t a l soluble nitrogen i n a l l three cuttings. The trend followed, resembles somev/hat that of t o t a l non-protein nitrogen. This i s p a r t i c u l a r l y true of the f i r s t cutting. Furthermore, the curves for sub-peptone show the trend of protein synthesis and degradation throughout the season. It i s very significant that i n the very early stages of growth ' sub-peptone should be at i t s highest le v e l thus indicating that, nitrogen intake is more rapid than protein synthesis. However, by the time flower buds have appeared i n the f i r s t cuttings, 1 sub-peptone has decreased to the lowest level obtained i n any part of the cycle, thereby showing that synthesis has been s u f f i c i e n t l y rapid to keep pace with nitrogen intake. Between the appearance of flower buds and the time when the plants are one-tenth i n bloom sub-peptone increases i n accordance with the ~ 66 -previously noted increases i n t o t a l soluble nitrogen and pep-tone nitrogen and the corresponding decreases i n soluble proteins and proteoses. This observation i s i n accordance with the findings of Thomas (192?), who showed that the formation •of blossoms i n the apple tree resulted i n marked increases in the amounts of the simpler forms of nitrogen i n the new growth which were derived at the expense of the "reserve" proteins previously accumulated i n the older branches© In the second cuttings, sub-peptone represents a much lower proportion of the soluble nitrogen during the early stages of growth than previously, thereby suggesting the p o s s i b i l i t y that synthesis had been very rapid during the period following the removal of the f i r s t cuttings. . An extremely high proport-ion of the soluble nitrogen i s present as sub-peptone at the time of early flowering however, thus substantiating the fore-going deductions relative.to bloom formation. In the t h i r d cuttings sub-peptone presents the same picture at the pre-bud stage as was shown i n the f i r s t cuttings. However, the relationship between budding and the early part of flowering i s the reverse of that previously: encountered.. This observation need not invalidate the assumptions made previously with regard to the need for an abundant supply of r e l a t i v e l y simple forms of nitrogen i n order to meet the requirements of blooming. On the other hand, i t suggests the p o s s i b i l i t y that an abundance of s l i g h t l y more complex compounds such as proteoses and peptones can meet this requirement. The fact that proteoses and peptones increase furnishes further evidence i n support of this supposition. - 67 -and Borsook i n terms of Maximum Total Soluble Nitrogen - In order that the exact nature of the processes of protein synth-esis and degradation might "be the more cle a r l y described, the .variations in the soluble nitrogenous fractions (W. & B.) obtained by calculations based on the data in Table I I and expressed i n percentage of the maximum t o t a l soluble nitrogen, are plotted i n Pig. 13. The results expressed i n this fashion apply only to the stages of growth from which the f i r s t cutt-ings were taken and do not include the results obtained from subsequent cuttings since they are incomplete with regard to stages of growth. Also the data relative to the seedling stage is omitted i n view of the fact that this material was obtained from a different planting. Consequently the conditions under which i t was grown would not be quite the same as those prevail-ing i n the case of the other samples. The curves for t o t a l soluble nitrogen, t o t a l non-protein nitrogen and sub-peptone show that synthesis and breakdown have alternated throughout the period under discussion. Synthesis has exceeded breakdown from pre-budding to budding and from the early part of flowering to f u l l bloom. Whereas from the time of budding to the early part of flowering and from f u l l bloom u n t i l maturity, breakdown overweighs synthesis. Consequently the curves f o r these fractions might be designated as "percentage synthesis-hydrolysis curves'1. In following the curve for soluble protein i t i s inter-esting to note that the peak of synthesis i s reached at the time FKU^jas-mieuTion OF yi.i iw F HITROOFN FP^CTIOMS QF W A V T F Y S * - 69 -of budding, which corresponds.very closely to the peak of vegetative growth. While from budding to maturity the trend is continually downward thus indicating a steady decrease i n anabolic processes. In so far as proteose and peptone are considered i t i s rather d i f f i c u l t to draw definite conclusions concerning their relationship to general metabolism throughout the season, i n view of the fact that they are present i n rather minute quantities. However, i t i s rather significant that the peak of both proteose and peptone coincides with the peak of great-est breakdown and that this i s reached at the next stage of growth after the peak of soluble proteins. The marked differences occurring between the different stages of growth studied i n this investigation suggest that further information might be gained concerning the nitrogen metabolism of the a l f a l f a plant i f a study of this nature were conducted on fresh samples collected at much shorter intervals throughout the l i f e history of the plants. In this way the exact gradations of synthesis and breakdown could be more cl e a r l y defined. The data presented i n Fig. 13 show, nevertheless, that the application of the method of Wasteneys and Borsook i n this study has made possible the presentation of a r e l a t i v e l y c l e a r l y defined picture of the trends of synthesis and break-down throughout the growing period of the plant. This i s / noteworthy i n view of the fact that the amounts of many of the fractions are extremely small and that the relationships exist-ing between them, at any one time, are much more complex than - 70 -those to be encountered at any stage i n the hydrolysis of pure proteins by defined enzymes* Acetic Acid Soluble Nitrogen - represents the nitrogen content of the water extract of a l f a l f a after the nitrogen compounds coagulable by heat and acetic acid have been removed. From Pigs. 14, 15 and 16, i t i s apparent the curves for this f r a c t i o n are at a much higher l e v e l than those for non-protein nitrogen (W. & B.) thus indicating that the removal of proteins by acetic acid, at the concentration used, is far from complete. In the second and t h i r d cuttings a r e l a t i v e l y constant relation-ship exists between the two curves, whereas i n the f i r s t cutt-ings there i s very l i t t l e agreement. No information whatever beyond that already provided by t o t a l soluble nitrogen can be derived from t h i s data. It should be pointed out, however, that the treatment with acetic acid was carried out primarily as the f i r s t step i n the amino nitrogen determination. Amino Nitrogen - This fr a c t i o n has already been defined as the nitrogen unprecipitated by phosphotungstic acid i n the acetic acid f i l t r a t e . However, i t should not be confused with amino nitrogen as determined by the method of Van Slyke (1912). The estimation of amino nitrogen as made i n t h i s study was carried out for the purpose of determining whether or not i t might serve as a short-cut anal y t i c a l method for the estimation of the amount of free amino nitrogen existing i n plant extracts. As mentioned previously, the procedure i s i d e n t i c a l with that used by Eagles and Sadler (l932)j which i n turn, i s based on the decomposition nitrogen of Orla-Jensen (1921). In discuss-FICl4-Dl5TRIBUT10n OF ACETIC A C V S O I . I N f ^ H ^ A M I D E N I T R O G E N . ! M - 74 ing the ripening of cheese Grla-Jensen defines the decomposit-ion nitrogen as, "the nitrogen of the protein decomposition products, or amino acids, which are not precipitated by phos-photungstic acid". Consequently this procedure was applied "to plant material to determine whether or not the results obtained would approximate those derived by the method of Yan Slyke ( 1 9 1 2 ) . Prom the curves portrayed i n Pigs. 1 4 , 1 5 , and 16 i t i s quite apparent that the amino nitrogen results show a considerable degree of i r r e g u l a r i t y . It should be pointed out that the amounts of this fraction obtained are consider-ably higher than those obtained by other workers in the application of the method of Van Slyke to plant material. Time did not permit of making Van Slyke determinations on this material consequently no comparisons can be drawn between the results of the two methods. The i r r e g u l a r i t i e s in the results obtained may be due in part to variations i n the effects induced by the prelimin-ary treatment with acetic acid. A certain amount of hydrolysis had apparently taken place during boiling because Nessleriz-ation of the f i l t r a t e s showed the presence of appreciable amounts of free ammonia whereas prior to this treatment only the merest traces were evident* Owing to the extreme complex-i t y of the material at various stages of growth the effects of this extremely miIQVhydrolysis would tend to vary consider-ably. Furthermore, Vickery ( 1 9 2 7 ) has shown that a very high degree of inconsistency exists with regard to the compounds - 75 -precipitated from plant extracts by phosphotungstic acid. This apparent lack of selective power on the part of phos-photungstic acid i s explained by him as being due i n part to the existence of certain complex interrelationships between ' the nitrogenous, and non-nitrogenous compounds present i n plant extracts. At any rate the results obtained i n this investigation tend to be i n accordance with those of Vickery and thereby suggest the need of further study before any definite conclusions may be drawn. Amide Nitrogen - The amide nitrogen of this investig-ation i s that nitrogen not precipitated i n the presence of phosphotungstic acid from the o r i g i n a l water extract of a l f a l f a . The preliminary treatment with dilute acetic acid i s omitted consequently the precipitation takes place under different conditions of concentration of the reagents. Poss-i b l y , in view of the methods of procedure followed, the amino and amide nitrogen results might be expected to approximate one another. They do not do so in every case, however, nor i s there any definite relationship between the amounts of these fractions at a l l stages of growth i n the various cuttings, Nevertheless, attention should be drawn to the fact that amide nitrogen i s present i n smaller amounts than amino nitrog-en at every stage of growth. Also, at the pre-bud and bud stages of growth i n the second cuttings the two fractions very nearly approximate each other i n the amounts obtained* At the seedling and pre-bud stages i n the f i r s t cuts amino nitrogen . does not greatly exceed amide nitrogen. - 76 Some importance might, in addition, be attached to the fact that, at the later stages of growth in both the f i r s t and second cuttings amino nitrogen i s present in much larger amounts than amide. A possible explanation i s that the compounds not precipitated by phosphotungstic acid i n the amide determination are of the same type as those formed during the processes of protein synthesis. Consequently, the proportion of these compounds would tend to decrease markedly throughout the later stages of growth. In the case of the amino determination, on the other hand, the conditions are such that a number of non-protein nitrogenous compounds, which would otherwise be removed, escape precipitation. Therefore i t i s possible that, i n the la t e r stages of growth, the more or less complex non-protein nitrogen compounds are of a type associated with the early phases of protein decomposition and consequently would be very readily decomposed by the mild hydrolysis with dilute acetic acid. Compounds of a correspond-ing degree of complexity formed as a result of synthesis would, in a l l probability, be of a s l i g h t l y different structure as well as being i n very intimate association with other non-nitrogenous products formed at the same time thus tending to render them impervious to the mild hydrolytic treatment. S B I M A R X A study has been made of the nitrogen distribution i n a l f a l f a hay cut at varying degrees of maturity throughout the growing period. For the determination of the soluble forms of nitrogen a departure has been made in applying to thi s study the method developed by Wasteneys and Borsook for the f r a c t i o n a l analysis of incomplete protein hydrolysates. In addition, the method evolved by Orla-Jensen and further developed by Eagles and Sadler, for the determination of amino and amide nitrogen has been employed. This was done with a view to determining whether or not the results obtain-ed would approximate those derived by the methods usually employed for the estimation of these fractions i n plant material. The data for t o t a l nitrogen shows a r e l a t i v e l y steady decrease throughout the growing period. Also, later cuts i n the season exhibit a d i s t i n c t l y higher t o t a l nitrogen content than e a r l i e r cuts at the same stages of growth. Total soluble nitrogen tends to exhibit considerable fluctuation throughout the growing period. Cuts made later i n the season show a progressive decrease i n the proportionate amounts of this fraction. P a r t i c u l a r l y s t r i k i n g is the f i n d -ing that, i n a l l cuttings, t o t a l soluble nitrogen tends to be present i n very large amounts during the early part of flower-ing. - 78 -Results obtained from the application of the method of Wasteneys and Borsook make possible the presentation of a r e l a t i v e l y c l e a r l y defined picture of protein synthesis and degradation throughout the growing period. This data further " strengthens the assumption that the period between bud form-ation and the commencement of flowering represents a very c r i t i c a l stage i n the l i f e history of the plant, Mb definite conclusions can be drawn from the amino and amide nitrogen results obtained i n this investigation. However, there are indications that more consistent results . might be obtained following a thorough t r i a l of the method. The possible relationships between nitrogen d i s t r i -bution and hay quality are also discussed. It i s quite evident that, from the standpoint of protein content, l a t e r cuts i n the season are superior to early cuts at the same stages of growth*, Gutting of hay at the f u l l bloom stage and later results i n a lowered percentage of t o t a l crude protein per ton of hay. The possible effects of the soluble nitrogenous constituents on hay quality are not so cle a r l y defined. However, since the amounts of each soluble constituent are so small i t i s probably safe to say that, i n d i v i d u a l l y , they do not exert any great influence on quality or feeding value. - 79 « ACOOILBDGBMEETS The author wishes to thank Dr. G. G. Moe for his support of the work described i n t h i s paper. Thanks are also due to Dr. D. G. 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