ROCZNIKI GLEBOZNAWCZE T. XXII, Z. 1, W ARSZAWA 1971

LESZEK CZUCHAJOWSKI, ALEKSANDER ERNDT

AMINO ACIDS COMPOSITION OF PROTEINACEOUS COMPONENTOF SOIL HUMIC ACIDS

Department of General Chemistry, University College of Agriculture, Kraków

HUMIC ACIDS STRUCTURE AND THEIR NITROGENOUS COMPONENTS

The organic m atte r of the soil is a complex of substances the composi­tion of which is determ ined in part by the p lan t and anim al residues added to the soil, and to a g reater extent, by the transform ations of these sub­stra tes through physical, chemical and biological means. Soil organic m at­te r contains most, if not all, of the natu ra lly occurring organic compounds. In addition to the in term ediary products of organic m atter breakdow n and secondary synthetic products of microbial action, there exists in the soil an accum ulation of the m ore resistan t products of decomposition — the soil humus.

The native organic fraction of soils is made up of a heterogenous m ix­tu re of polymerized arom atic molecules, polysaccharides, bound amino acids, uronic acid polymers, and various organic phosphorus compounds [27]. By definition, hum us is a complex m ix ture of amorphic and colloidal substances arising from modified plant m aterials and synthesized m icro­bial tissue.

The characterisation of hum us is far from complete, and concepts con­cerned w ith its form ative processes are, to a large extent, speculative. Early theories postulated th a t lignin complexed w ith protein of microbial origin provided the m ain source of hum ic substances in soil. Humic acids w ere found to yield degradation products identical w ith those obtained from lignin. The sim ilarity betw een the arom atic s truc tu re of lignin and that found in hum ic acids, and the rela tive resistance of lignin to enzym a­tic breakdow n m ay be said to be in good agreem ent w ith the lignin theory of hum us form ation [45].

L. Czuchajowski, A. Erndt

More recent studies, however, suggest th a t m any compounds of non- lignin origin m ay also be a source of arom atic “s truc tu ra l clusters” . Mi­cro-organisms are able to give rise to compounds of an arom atic or hetero- arom atic natu re through the conversion of m any diverse organic com­pounds including carbohydrates [34]. Such arom atic struc tu res can be oxi­dized and condensed w ith amino acids or peptides w ith the form ation of substances sim ilar to those found in the soil humic acid fraction. It appears at the present tim e th a t the “nuclei” of hum ic acids arise both from a lte ­red lignin and tann in compounds and through the synthesis of arom atic compounds by micro-organisms. It seems probable th a t m any products of synthesis and m icrobial m etabolism such as amino acids, peptides, am i- nosugars, nucleoproteins, purine and pyrim idine bases, uronic acids, orga­nic acids, alcohols, etc., participate in the form ation of the functional groups of hum us.

The chem istry of soil organic m atte r is best studied in the absence of inorganic m atrix of soil and therefore the organic m atte r m ust first be dissolved by an aqueous or nonaqueous ex tractan t. A t present available extraction procedures are ra th e r empirical, and the complete extraction seems to be an impossibility. On the other hand, most extraction procedures probably produce artifacts, organic m atte r extracted from the soil m ay be very d ifferen t from th a t which exists in the soil. Oxidation, polym eri­zation and photochem ical reactions can change to some ex ten t the paren t m aterial during extraction.

Extraction is m ade difficult by the adsorption of organic m atte r on m ineral constituents of the soil. Due to the ir great surface area and p re­ponderance of reactive adsorption sites, clay m inerals lower the extraction efficiency. It has been shown [21] th a t polar compounds and non-ionic compounds are adsorbed on clay, particu larly on the basal surfaces of ex­panding clays. A liphatic molecules are oriented w ith the plane of their chains both parallel and perpendicular to the clay surface. Some organic molecules are held to the clay surface through С—H " '0 bonds [3, 12, 29], although any adsorbate capable of supplying electrons to the incom plete p orbitals of adsorbed or lattice alum inium could be adsorbed as a carbon­ium ion [25]. Detailed study has shown [11] th a t exposed lattice alum in­ium or iron are involved in the adsorption of carbohydrates on clay. Organic cations and polycations, such as proteins, below the isoelectric point are adsorbed on clay by a cation-exchange mechanism. These cou- lombic forces are probably supplem ented by van der W aals forces and С—H ""0 bonds betw een the organic molecule and the clay m ineral su r­face. The use of a basic ex tractan t rem ove adsorbed am photeric com­pounds. An increase in basicity renders the organic m atte r electronegative and prevents extensive adsorption by coulombic repulsion forces [32].

Amino acids composition... 77

In addition to the quan tity of organic soil m atte r held to the clay su r­face, there are indications th a t significant am ounts of organic molecules in soil and sedim ents occur in in tim ate association w ith clay [40]. It has been suggested th a t amino acids and peptides are adsorbed on in terla- m ellar surfaces of clay m inerals. Result of X -ray diffraction studies have shown th a t there is an apparent “contraction” of the thickness of amino acids and peptides by adsorption on in terlam ellar positions of m ontm o- rillonite, indicated th a t the adsorbed molecules are “keyed” into hexagonal holes [19]. Amino acids or peptides bound to clay in this m anner would be particu larly difficult to rem ove by chemical or biological agents. The adsorption of amino acids by clay provides a partial explanation for their ability to survive in sedim ents and sedim entary rocks over geologically long periods of tim e [39].

A nother factor w hich affects the solubility of organic m atte r is the for­m ation of salts and gels w ith m etal ions. The organo-m ineral colloids in soil are a complex m ix ture of floccules and gels [44], the stab ility of which depends on the pH and oxidation-reduction status of the soil [1]. These organom ineral gels are transform ed into w ater-soluble hydroxy-com plexes by partia l hydrolysis in a slightly alkaline medium. In a strongly alkaline m edium they are com pletely hydrolysed [26].

The rem oval of the organic phase by solvent extraction of the soil gives a large num ber of relatively simple compounds of know n structu res such as carbohydrates, carboxylic acids, esters, amino-acids, glycosides and or­ganic bases. The rem ainder of the soil organic m atte r consists of a m ix tu re of am orphous brow n or black products referred to as “hum us”, and par­tially decomposed p lan t and anim al residues. “H um us” has been separated into fractions differing in solubility in w ater, alkalis, acids and organic solvents, bu t in view of the heterogeneity of the fractions d ifferen t term i­nologies have been used by d ifferen t authors. In the course of the present work, the term “hum ic acids” is used for th a t part of hum us which is soluble in 0.5 N sodium hydroxide and precipitated by acidification of the alkaline extract. Such a definition has been w idely used, although O d e n [33] rem oved the alcohol-soluble part of the precipitate as “hym atom e- lanic acids” ; it is, however, doubtful w hether such a sub-division is desi­rable [2]. The term “fulvic acids” relating to the portion of “hum us” which rem ains in the aqueous liquor afte r the acidification of the alkaline extract, is still reta ined by m any w orkers although this fraction includes polysaccharides, peptides, amino acids and relatively simple phenols as w ell as compounds of unknow n structure.

The composition of hum ic acids is determ ined by the source and isola­tion technique. E lem entary analyses of products isolated from various soils have given values ranging for С 49-62%, H 3-6% . N 0.4-5.0%, and

78 L. Czuchajowski, A. Erndt

m olecular w eights from 1000 to 300,000 are reported [2]. D egradation of hum ic acids involving fusion w ith alkali [22], oxidation w ith n itric acid [42], perm anganate [30], alkaline nitrobenzene [31], or copper oxide [18] gave small yields of phenols such as catechol, resorcinol, and phloroglu- cinol, aldehydes including p-hydroxy-benzaldehyde, vanillin, syringalde- hyde and acids such as p-hydroxybenzoic, vanillic, protocatechuic, vera t- ric and isohemipinic acids.

Recent study [4, 24] have shown th a t some of the phenols, based upon resorcinol or phloroglucinol structures w ere a ttribu ted to flavonoid p re­cursors, others based upon catechol types w ere regarded as lignin-derived units. It was then concluded th a t besides lignin, flavonoids and possibly other natu rally occurring polyphenols m ay be involved in the form ation of humic acids, as suggested earlier by T r u s o v [43].

Infrared absorption bands of humic acids in the region of 1600 cm -1, 1510 cm -1 and 840 cm “1 are correlated w ith the presence of arom atic rings [35]. In particular, rings of this type have been postulated in a quantum chem istry in terp re ta tion of in frared absorption changes w hich appeared by pyrolysis of humic acids and of model arom atic compounds having OH groups [6, 10]. More recent experim ents [5] on the degradation of humic acids using zinc dust distillation at 500°C in a stream of hydrogen have proved the presence of arom atic nuclei in the humic acid macromolecule. A nthracene and 2,3-benzofluorene w ere isolated in crystalline pure form from the destillate. A combination of UV spectrom etry, vapour phase chrom atography and mass spectrom etry provided convincing evidence of the presence of naphthalene, 1- and 2-m ethylnaphthalenes and a wide range of o ther arom atic hydrocarbons in the distillate, including pyrene, perylene, 1,2-benzopyrene, and coronene, together w ith homologues, and also the presence of homologous acridines and 1,2- or 3,4-benzoacridines. In a subsequent experim ent oxidation of hum ic acids w ith perm anganate followed by decarboxylation in quinoline and C uS 04 gave anthraquinone, m ethylanthraquinones, 2-m ethylnaphthalene, fluorenone, xanthone, m e- thylxanthones and the hydrocarbons C13H12, C14H10 and Ci5H14. Even though the products from the zinc dust distillation m ay arise from secondary re ­actions this is im probable in the case of products from the perm anganate oxidation. Consequently, it is believed tha t the existence of a polynuclear arom atic struc tu re in humic acids has been established.

Electron param agnetic resonance spectra have shown [37] th a t soil hum ic acids contain stable organic free radicals of the order of 1018 spin/g. These radicals are exceptionally stable w ith respect to tim e and chemical attack. The samples have shown no detectable decrease in spin concen­trations over a period of th ree years. Extensive acid hydrolysis of humic acids only increased the free radical content in the hydrolysate; this same

Amino acids composition... 79

behaviour was encountered w ith high tem pera tu re oxidation, w hereas the reduction of hum ic acids w ith sodium m etal and lith ium alum inium hy ­dride caused only a small decrease in spin concentration.

It would appear th a t free radical species are an in tegral p a rt of the natu ra l humic acids macromolecules and the radical s truc tu re is e ither shielded by the polymeric netw ork or stabilized by delocalization of the unpaired electrons over a suitable arom atic system.

As a significant increase in spin concentration has been observed for the samples w hen they w ere converted to the sodium salts, it is assum ed th a t humic acids contain sem iquinone radicals coexistent w ith quinhy- drone moieties, probably derived from ortho or para benzoquinones.

It is characteristic tha t our previous study [8, 9] on the origin of the absorption band near 1600 cm -1 in the infrared spectra of humic acids have postulated the presence of hydroxyphenyl-quinones and of some simple condensed hydroxyarylquinones “in form of semiquinones and hydro- quinone-quinone charge-transfer complexes, as the s truc tu ra l units in the humic acids macromolecules.

The general s truc tu re (I) has recently been advanced [5] in which hu ­mic acid is a complex consisting of a polycyclic arom atic “core” attached to polysaccharides, proteins, relatively simple phenols and metals.

Carbohydrates ----------- Polypeptides

Polycyclic x aromatic core

Phenolic acids ------------- Metals

(I)

Most of the carbohydrates are probably present as polysaccharides. F o r s y t h [16] showed th a t boiling w ater rem oved from hum ic acids de­rived from a varie ty of sources, about 20% as a polysaccharide m ix ture yielding on acid hydrolysis glucuronic acid, galactose, glucose, mannose, arabinose, xylose and ribose. Rare 4-O-m ethyl-D -galactose and 2-O-me- thyl-L -rham nose have been isolated [13] from a peat polysaccharide.

The rem oval of carbohydrates and phenolic acids by m eans of cold or hot w ater is not necessarily evidence in favour of physical adsorption; readily hydrolysable depsidic, ester and glycosidic linkages are frequently encountered in other fields, e. g. the gallo- and ellagitam ins [20], and the hydrolysis of hum ic acids would probably be facilitated by the presence of carboxyl or o ther acidic groups in the molecule. There is no doubt th a t polysaccharides of the hum ic acid fraction of the soil play an im portant

80 L. Czuchajowski, A. Erndt

role in the cation-exchange capacity, carbon metabolism, and complexing of m etals in soil.

The mode of a ttachm ent of the proteinaceous constituents is still uncertain. However, m any authors [28, 41] considered th a t the stability tow ards chemical and m icrobial a ttack of the peptides was an indication th a t they are an in tegral part of the hum ic acid molecule. The stability m ay be caused by chemical combination of the peptides w ith quinone groups by reaction sim ilar to those frequen tly postulated in earlier theories of tanning.

Studies on model substances (II, III, IV) have shown [5, 14]

ОR y II R = —NH • CH2 • CO • OC2H 5

Y ) III R = — CH3II \ n h • CH2• CO• OC2H5 IV r = _ o c h 3о

th a t all th ree compounds lost glycine on hydrolysis w ith 6 N HC1 just as the nitrogen was rem oved from humic acids by hydrolysis. S im ulta­neously, in all th ree cases black am orphous substances w ere produced, presum ably by fu rth e r condensation of the hydroxy-p-benzoquinones resulting from the rem oval of the glycine. However, all th ree compounds investigated are unlikely to lose their nitrogen by trea tm en t w ith w ater, and the rem oval of a small am ount of peptide-like substances by extrac­tion w ith cold or hot w ater suggests either physical attachm ent or a lte r­natively, attachm ent by hydrogen bonding betw een the imide groups of the peptide and phenolic groups (structure V or VI).

HO -> H—N—C-i i и

^ R O

V

xRfH—N<

-Q / > \ r <-o ^

VI

Some nitrogen in hum ic acids is stable against hydrolysis w ith 6 N HC1 and unaffected by m éthylation and acétylation [15, 23] and m ay be present in heterocyclic form.

The present study was undertaken to determ ine quantitatively the amino acid composition of the peptide and protein content of soil hum ic acids, its characteristic response to the conditions of hydrolysis, and to investigate the relation to the amino acid composition of the soil and green p lan t species.

Amino acids composition... 81

EXPERIMENTAL

As a resu lt of collaboration w ith the D epartm ent of Soil Science, U niversity College of A griculture, Cracow, it has been possible to exam ine humic acids extracted from the following soils.

Soil I; source: A gricultural Experim ent Station (RZD) Prusy, field B, profile located ca. 300 m from the road. P aren t rock: loess; p lan t cover: arable field prepared for experim ental work, sample from horizon A x (В ) 25-30 cm, brow n-grey fine sand w ith hum us streaks, slightly moist, crum b structure, loose, no roots, pH 7.0; soil type: proper brow n soil, soil family: form ed on loess, tex tu ra l group: fine sand;

Soil II; source: peat meadow, Szarów. P aren t rock: lowmoor fen tend­ing to transition moor, on M iocenian clay; p lant cover: Ranunculus sp., Taraxacum palustre , Plantago lanceolata, Beilis perennis , Poa annua , sample T2, 51-126 cm, brow n peat slightly decomposed, wet; soil type: peat soil, soil family: form ed on lowmoor fen w ith underlying Miocenian clay; peat species: A ldersedge peat.

ISOLATION OF HUMIC ACIDS

According to the standard procedure [2], the a ir-dried soil sam ple was defatted w ith a m ix ture of equal volumes of ethyl alcohol and benzene, and dried at 90°C. The defatted soil (20 g) and 250 cc of 0.2 N NaOH, after standing at room tem peratu re for 18 hrs w ith occasional shaking, w ere centrifuged. The supernatan t liquors w ere filtered through a sin tered glass funnel (No. 4) and the filtra te was acidified w ith 0.2 N HC1 to pH 1. For collection of the hum ic acids a m odified F o r s y t h and F r a s e r ’s technique [17] was used, in which the acidified m ix ture was centrifuged, the liquor decanted and the moist precipitate was frozen to — 50°C for 12 hrs. Then, it was allowed to thaw and the solid collected on a sintered glass funnel (No. 4), washed un til free from chloride, and dried at 90°C.

The yield of hum ic acids ex tracted from the soil I was 0.2*Vo, and from the soil II — 26’°/o, respectively. The resu lts of elem ental analyses are included in Table 1.

ACTION OF HYDROCHLORIC ACID ON HUMIC ACIDS

F i r s t h y d r o l y s i s : The sample of 0.2 g of hum ic acids and 20 cc of 6 N HC1 w ere placed in a glass tube which was then sealed under nitrogen. The hydrolysis was carried out by heating the tube content in an oven at 110°C for 20 hr. Then, the tube was opened, the liquor filtered through a sin tered glass funnel (No. 4) and the solid w ashed w ith th ree portions of 25 cc dist. w ater. The combined filtra te was evaporated to

6 Roczniki G leboznaw cze, T. XXII, z. 1

82 L. Czuchajowski, A. Erndt

T a b e l a 1Elementary a n a ly s is o f s o i l humic a c id s and o f th e res id u es

a f t e r th e ir h y d ro ly s is

Elementary сc a p o s it io n

/% /

Decrease o f n itro g en in % o f i t s content in :

No. M ateria l

С H Nparenthumicac id s

resid u ea f te rf i r s thydro­l y s i s

1 Humic a c id s derived from th e brown lo e s s , Prusy, I 52 ,51 4 ,8 4 2 ,77

2 Residue a f t e r f i r s t h y d ro ly s is1 ,53

1 ,36

> 4 %

3

o f I

Residue a f t e r second h y d ro ly s is 4 5 ,31 3 ,5 8>11%

4 Humic a c id s d erived fron the peCst meadov/, Sżarów, I I 53 ,49 5 ,3 4 4 ,0

5 Residue a ft e r f ir s t ' h y d ro ly s is o f I I

Residue a f t e r second h y d ro ly s is

57 ,97

59,42

4 ,7 6

4 ,3 9

o in> 4 S £

6 1 ,23>3C?&

T a b e l a 2Amino a c id com position o f p ro te in aceou s component o f s o i l humic a c id s; the data

exp ress a percentage n itro g en co n tr ib u tio n o f each amino ac id in th e t o t a l n itro g en content o f a l l amino a c id s recovered

Humic a c id s from th e brown lo e s s s o i l , Humic a c id s from the peat meadow s o i l ,

Amino Prusy, / I / Szarów, / I I /

acidF ir s t

h y d ro ly za tes ,4

Secondh y d ro ly za tes ,

ZB

F ir s th y d ro ly za tes ,

Gecondh y d ro ly za tes ,

IXb

LYS 9 ,9 6 ,8 5 ,2 1 5 ,8 24 ,0 8 ,3 1 0 ,4 9 ,4 2 2 ,0 12 ,0HIS 4 ,8 2 ,3 1 .3 6 ,2 X 4 ,0 3 ,0 3 ,6 10 ,5 X

ARG 8 ,8 4 ,0 3 ,5 16 ,0 X 6 ,6 5 ,4 3 ,5 13 ,9 X

ASP 1 3 ,4 12 ,2 14 ,3 6 ,2 6 ,6 13,2 9 ,9 14 ,2 7 ,7 8 ,3THR 4 ,0 4 ,8 6 ,4 2 ,6 3 ,4 5 ,2 4 ,5 6 ,0 3 ,0 3 ,0SER 3 ,4 4 ,7 5 ,7 2 ,6 4 ,0 4 ,1 4 ,4 4 ,9 3 ,1 4 ,3GLU 9 ,3 10 ,7 9 ,4 6 ,1 8 ,3 8 ,9 9 ,0 10 ,6 8 ,0 10 ,2PRO 6 ,4 9 ,5 8 ,1 4 ,7 7 ,1 5 ,0 3 ,1 8 ,0 4 ,5 6 ,9GLY 8 ,3 10 ,1 1 3 ,4 6 ,2 8 ,3 10 ,1 1 1 ,4 12 ,2 9 ,2 11 ,1ALA 8 ,3 10 ,1 9 ,6 4 ,4 6 ,1 12 ,0 10 ,4 11 ,5 5 ,9 8 ,0VAL 6 ,7 6 ,2 6 ,7 9 ,3 8 ,6 6 ,8 6 ,2 8 ,6 11 ,3 10 ,6MET 0 ,8 0 ,4 0 ,4 0 ,2 0 ,9 0 ,4 0 ,3 0 ,5 0 ,0 5 tr a c esI LEU 3 ,7 3 ,5 3 ,8 6 ,2 7 ,0 3 ,7 3 ,8 5 ,3 7 ,5 8 ,5LEU 6 ,7 6 ,8 6 ,7 6 ,4 8 ,2 6 ,3 6 ,0 8 ,0 8 ,1 9 ,9TYR 1 ,7 1 ,5 1 ,4 2 ,6 2 ,1 1 ,8 1 ,1 1 ,1 2 ,0 2 ,1PHEN 3 ,7 5 ,4 3 ,9 4 ,6 4 ,8 4 ,0 5 ,6 3 ,8 4 ,9 5 ,0

Uniden­t i f i e d

Long c o l . , b u ffe r I I : ca. 53 m in ., ca 75 min.

absentLong c o l . , b u ffe r I I : ca 53 m in ., ca 75 min ca 75 min.

Amino acids composition... 83

dryness a t 40-45°C and used for determ ination of amino acids. The solid collected on the funnel was dried in vacuum to constant weight, analyzed for C, H, N content and retained for nex t hydrolysis.

S e c o n d h y d r o l y s i s : The dried sample (0.2 g) of the solid residue after first hydrolysis and 20 cc of 6 N HC1 w ere placed in a glass tube, sealed under nitrogen and trea ted as described above. The evaporated filtra te was used for the determ ination of amino acids. The rem aining residue was analysed for C, H, N, content.

Both hydrolysis procedures w ere carried out on replicate samples and all determ inations repeated twice.

The resu lts in Table 3 show the m ean values of two or th ree separate estimations; these are presented in Table 2.

AMINO ACIDS ANALYSIS

The analyses w ere carried out on the Beckm ann-Spinco 120B amino acids autom atic analyser, fitted w ith two columns. The longer one, 50 cm long and 0.9 cm in diam eter, filled w ith Custom Spherical Resin Type AA-15 (minimal 8'°/o crosslinked sulphonated styrene copolymer), was eluted in a standard way w ith 0.2 M citric buffer at pH 3.28. In about 150 m inutes buffer I was replaced by citric buffer II, pH 4.25. The short column, 10 cm long and of the same diam eter, filled w ith AA-27 resin, was eluted w ith citric buffer pH 5.28. The investigated samples w ere in­troduced on columns afte r dissolving them in 0.1-2 cc of the buffer. The concentration order of the solution derived from the in tensity of chrom a­tograms.

The reproducibility of m axim a positions of the calibration m ix ture components was perfect as long as the same solution of n inhydrin reagent and of the buffers w ere used throughout the analysis. However, in analyses carried out w ith new ly prepared portions of n inhydrin reagent and of the buffers, the positions for individual amino acids varied: for the compounds appearing w ith in the first 70 m inutes (long column separation) the change was w ith in the range of 2 min., for those appearing la ter w ith in the range of 4 min. By the short column separation the differences w ere in the range of 2 min., except for ARG which was revealed at in tervals of 4-5 min.

Elution tim e for the amino acids was as follows; short column: LYS (ORN) 27, HIS 37, ARG 68; long column, I buffer: ASP 45, THR 53, SER 56, GLU 64, PRO 74, GLY 88, ALA 95, ABUT 110, VAL 128; II buffer: ILEU 8-12, LEU 16, TYR 34, PHE 41 m inutes. Q uantitative calculations w ere m ade by the standard m ethods using the calibration curves as reference.

6*

Amino acid com position o f prote in aceou s component o f s o i l humic ac id s; mean v a lu es o f n itrogen co n tr ib u tio n from Tab. 2 , compared w ith the v a lu es g iven fo r s o i l and p lan t p ro te in h y d ro ly za tes ,

/3 6 ,3 8 ,4 8 / and /4 6 ,4 7 /

T a b e l a 3 oo

Aminoacid

Humic ac id sS o i l s from

Leaves o f Phaseо lu s v u lg a r is ,

p ro te in fr a c t io n s

/4 7 /

Lucerne /4 6 /

Grassp ro te in s

/4 6 /

Prusy / I / Szarów / I I /

f i r s thydrol.

4

second h y d ro l.

*B

f i r s thydrol.

П А

secondhyd rol.

K B

Lacombe/3 6 /

Flanagan,s i l tloam,/3 8 /

H o y tv i l lc , c la y loam, /4 8 /

wholep ro t.

cyto­plasm.p ro t.

A В

LYS 7 ,3 1 5 ,4 9 ,4 17 ,0 15.7* 13 ,4 1 4 ,0 9 ,5 10,8 9 ,4 9 .4 9 .4

HIS 2 ,8 6 ,2 3 ,5 10 ,5 2 ,4 * 1 .7 2 .5 5 ,8 4 ,4 5 ,1 5 .0 4 .7

ARG 5 ,4 16 ,0 5 ,2 13 ,9 4 ,1 * 4 ,9 2 ,8 16 ,0 11.5 16 ,6 15.7 14 ,4

ASP 13,3 6 ,4 12 ,4 8 ,0 13 ,5 6 ,3 13 ,6 8 ,4 10 ,0 7 ,8 8 ,0 8 .1

THR 5 ,1 3 ,0 5 ,2 3 ,0 9 ,1 8 ,4 6 .7 4 ,6 4 ,7 4 ,2 4 ,6 4 ,6

SER 4 ,6 3 ,3 4 ,5 3 ,7 5 ,6 8 ,6** 7 .6 5 ,2 6 ,4 4 ,2 3 ,9 4 ,8

GLU 9 ,8 7 ,2 9 ,5 9 ,1 9 ,7 7 ,9 9 .2 9 ,0 9 .7 7 ,7 8 ,6 7 ,6

PRO 8 ,0 5 ,9 7 ,0 5 ,7 4 ,3 5 ,6 4 ,1 4 ,3 5 ,0 4 ,5 4 ,0 4 ,6

GLY 10 ,6 7 ,3 11,2 10 ,2 13 ,2 12,2 1 0 ,6 7 ,9 7 ,8 7 ,5 6 ,7 7 ,9

ALA 9 ,3 5 ,3 11.3 7 ,0 6 ,3 10,7 12 ,2 6 ,9 7 ,4 7 ,1 7 ,5 8 ,0

VAL 6 ,5 8 ,9 7 ,2 11 ,0 6 ,0 6 ,5 6 ,9 5 ,4 6 ,0 5 ,9 5 ,1 6 ,4

MET 0 ,5 0 ,4 0 ,4 tr a c e s not given 1 .2 1 .3 1 .3

I LEU 5 ,7 6 ,6 4 ,4 8 ,0 3 ,2 4 ,5 2 ,5 3 ,8 3 ,9 4 ,4 5 .1 4 .2

LEU 6 ,7 7 ,8 6 ,8 9 ,0 3 .7 5 ,9 4 ,4 7 ,4 7 ,0 7 ,5 7 .5 7 ,2

TYR 1 .5 2 ,4 1 .3 2 ,1 1 .8 1 .6 0 ,9 2 ,6 2 ,3 2 ,8 3 .1 2 ,8

PHEN 4 ,3 4 ,7 4 ,5 5 ,0 2 ,7 1 .8 2 ,1 3 ,6 3 ,2 3 .9 4 .4 4 ,0

* / not g iven in / 3 6 / , supplemented by the authors*, * * / not given /3 8 / , supplemented by the authors

L. Czuchajow

ski, A. Erndt

Amino acids composition... 85

RESULTS AND DISCUSSION

The amino acid contents of acid hydrolyzed hum ic acids derived from the brow n loess soil (I, Prusy) and from the peat meadow soil (II, Szarów) are shown in Table 2. The data express a percentage nitrogen contribution of each amino acid in the to tal nitrogen content of all amino acids recovered. W ithin individual amino acids which are typical constituents of protein preparations tryp tophan was not estim ated because of its destruction under the conditions of hydrolysis employed. On the other hand, the results presented in Table 2 showing the tim e of elution of amino acids from the column indicate the presence of some unidentified com­ponents giving a positive reaction w ith ninhydrin. There are two com­pounds appearing in ca. 53rd and 75th min. of elution w ith buffer II from the long column which m ay be regarded as glucosamine and galactose- amine.

P resentation of analytical results included in Table 3 shows clearly the relation betw een amino acid composition found in hum ic acids and th a t of p lan t protein preparations. It seems also useful to point out certain considerations regarding the relationship betw een amino acid composition determ ined in the present w ork and the amino acid content of acid hydrolyzed soils reported in the lite ra tu re [32, 36, 38, 48]. The comparison of the amino acid composition in humic acids w ith th a t found in protein hydrolyzates from the leaf of Phaseolus vulgaris [47] is w orthy of special note because of an iden tity of the hydrolysis procedure and of the tech­nique of amino acids analysis.

In order to avoid inequality of samples associated w ith heterogeneous soil m aterial, each hum ic acids sample to be hydrolyzed consisted of several hum ic acids portions ex tracted either from the brow n loess soil (I, Prusy) or from the peat soil (II, Szarów).

In Table 1 are given the results of elem ental analyses of humic acids before hydrolysis and afte r the first and the second trea tm en t w ith 6 N hydrochloric acid. It can be seen tha t the first hydrolysis lowers the nitrogen content in the residue almost equally in hum ic acids I (Prusy) and in hum ic acids II (Szarów). The decrease is 45% in I and 48% in II, respectively, even though the paren t humic acids before hydrolysis differed m arkedly in their nitrogen content (2.77% N in I, 4.0% N in II). The second hydrolysis causes a differentiation in the release of nitrogen from the residue afte r the first action of 6 N HC1; the values are 11% and 30'% of the nitrogen present before the second treatm ent.

No large differences in amino acids content of the first hydrolyzates of hum ic acids I and II w ere found, although, at this stage of hydrolysis the content of alanine was 11.3% (II) and 9.3% (I). Differences in amino acids content in these hydrolyzates w ere w ith in the range of amino acid

86 L. Czuchajowski, A. Erndt

distribution in protein preparations of cytoplasmic and chloroplast frac­tions rem oved from the leafs of Phaseolus vulgaris [47].

L arger differences in the content of individual amino acids w ere found in the second hydrolyzates of I and II. The contents of LYS, HIS, ARG, ASP, GLU, GLY, ALA and VAL expressed as a percentage of the to tal amino acid nitrogen differed by m ore than 1.5'%. However, a certain regu larity of variations in amino acid content in the first and the second hydrolyzates of humic acids (II and I) derived from both types of soil is notew orthy. As the action of 6 N HC1 proceeded the content of LYS in the hydrolyzates expressed as a percentage of to tal amino acid nitrogen increased by ca. 8% (II) and 6% (I), th a t of HIS by 7% and 3%, ARG 9% and 11%, VAL 4% and 2%, ILEU 4% and 3%, respectively; the content of ASP decreased by 4% and 7%, and th a t of ALA was lower by 4% and 4%, respectively. In fact, these differences w ere associated w ith all analysed basic amino acids and does not refer to the neu tra l amino acids having cyclic fragm ents and hydroxyl groups.

It is of particu lar in terest th a t the amino acid composition of the first hydrolyzates of both humic acids (I and II) rem inds m uch m ore of the distribution of amino acids in p lan t protein of the aboveground p a rt of Phaseolus vulgaris [47] and of lucerne and grass [46], than it does the amino acid composition of second hydrolyzates. In the latter, only the contents of ARG, ASP, PRO and ALA m ay be related to the p lan t protein composition.

It seems also in teresting to compare the results of the present work concerning the amino acid composition of proteinaceous constituents of humic acids w ith those of acid hydrolyzed soils reported in the lite ra tu re [36, 38, 48], although some correction is needed for the presence of small quantities of free amino acids detected in soil [7]. D ifferent conditions of hydrolysis and analytical procedure m ay also influence the results. Nevertheless, the im pression is th a t the content of eight amino acids (LYS, HIS, ARG, ASP, GLU, GLY, VAL, ILEU) in the first hydrolyzates of humic acids is com parable w ith th a t found in hydrolyzed soil. W ith respect to the content of PRO and LYS soil hydrolyzates are sim ilar to the second hydrolyzates of humic acids.

Obviously, conclusions of an over-all na tu re requ ire m ore detailed study on hum ic acids derived from a w ider range of soil types. On the basis of the present results an assum ption can be m ade that: soil humic acids contain two m ain proteinaceous fractions differing in amino acid composition, or there is a difference in the susceptibility to hydrolytic cleavage of peptide bonds in the amino acid sequence of proteins and peptides which resu lts in two “fractions” having different amino acid com­position. In any case, amino acid d istribution in both “fractions” seems to

Amino acids composition.., 87

be representative of proteinaceous constituents of soil humic acids. The m ore easily hydrolyzed components appear to be sim ilar to p lan t protein.

It is difficult to neglect the possibility of decyclization of N-heterocyclic non-proteinaceous components of hum ic acids followed by splitting them into basic and chainbranched amino acids, on prolonged hydrolysis. This would be consistent w ith the increase of VAL and ILEU content found in the second hydrolyzates. Such reactions would be expected to contribute to the differentiation of amino acid content in the second hydrolyzates of hum ic acids derived from differen t soils.

The authors are indebted to Miss Zofia Pasternak for assistance in the laboratory work.

REFERENCES

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[2] A t h e r t o n N. М., С r a n w e 11 P. A., F l o y d A. J., H a w o r t h R. D.: Tetrahedron, 23, 1967, 1653.

[3] В r a d 1 e у W. F.: J. Am. Chem. Soc., 67, 975, 1945.[4] B u r g e s N. A., H u r s t H. M., W a l k d e n B.: Geochim. et Cosmochim.

Acta, 28, 1964, 1547.[5] C h e s h i r e M. V., C r a n v e i l P. A., F a l s h a w C. P., F l o y d A. J.:

Tetrahedron, 23, 1669, 1967.[6] С z u с h a j o w s k i L.: Roczniki Chem., 41, 237, 1967.[7] C z u c h a j o w s k i L., B a r u t o w i c z Z.: Bull. Acad. Polon. Sei., sér. sei.

biol., 18, no. 9, 1970.[8] C z u c h a j o w s k i L., E r n d t A.: Roczniki Chem., 43, 1969, 1451.[9] C z u c h a j o w s k i L., E r n d t A.: Bull. Acad. Polon. Sei., sèr. sei. chim., 18,

1970, 35.[10] C z u c h a j o w s k i L.: Roczniki Chem., 41, 1967, 593.[11] D e S. K., R a s t o g i R. C.: Z. Pflanzenernähr. Düng. Bodenk., 98, 1962, 121.[12] D e u e 1 H.: Trans. Intern. Congr. Soil Sei. 7th congr., 1, 1960, 38.[13] D u f f R. B.: J. Sei. Food Agr., 3, 1952, 140.[14] F i s c h e r E., S c h r ä d e r H.: Ber. Dtsch. Chem. Ges., 43, 1910, 525.[15] F o r s y t h W. G. C.: J. Agr. Sei., 37, 1946, 132.[16] F o r s y t h W. G. C.: Biochem. J., 41, 1947, 176; 46, 1950, 141.[17] F o r s y t h W. G. C., F r a s e r G. K.: Nature (Lond.), 160, 1947, 607.[18] G r e e n G., S t e e 1 i n к C.: J. Org. Chem., 27, 1962, 170.[19] G r e e n l a n d D. J., L a b y R. H., Q u i r k J. P.: Trans. Faraday Soc., 88,

1962, 829.[20] H a s la m E.: Chemistry of Vegetable Tannins, Acad. Press, Lond. 1966, pp. 92,

126, 129.[21] H o f f m a n R. W., B r i n d l e y G. W.: Geochim. et Cosmochim. Acta, 20,

1960, 15.[22] H o p p e - S e y l e r F.: Z. physiol. Chem., 13, 1889, 66.[23] H o s a d a K., T a k o t a H.: Soil and Plant Food, 3, 1957, 197.[24] H u r s t H. M., B u r g e s N. A., L a t t e r P.: Phytochemistry, 1, 1962, 227.

88 L. Czuchajowski, A. Erndt

[25] J u r i n a к J. J., V о 1 m a n D. H.: J. Phys. Chem., 63, 1959, 1373.[26] K a w a g u c h i K., K y u m a K.: Soil and Plant Food, 5, 1959, 54.[27] K o n o n o w a M.: Substancje Organiczne Gleby, ich Budowa, Własności i Me­

tody Badań, PWRL, Warszawa 1968.[28] L a d d N. J.: Austr. J. Biol. Sei., 17, 1964, 153.[29] M a c E w a n A. F.: Trans. Faraday Soc., 44, 1948, 349.[30] M o r r i s o n R. I.: Chem. and Ind., 1955, 231.[31] M o r r i s o n R. I.: J. Soil Sei., 9, 1958, 130; 14, 1963, 210.[32] M o r t e n s e n J. L., H i m e s F. L.: in F. E. Bear “Chemistry of the Soil”,

Reinhold Publish. Corp., 2nd ed., N. Y., 1965, p. 209.[33] O d e n S.: Kolloidchem. Beihefte, 11, 1919, 75.[34] P i n c k L. A., D y a l R. S., A l l i s o n F. E.: Soil Sei., 78, 1954, 109.[35] S c h a r p e n s e e l H. W., A l b e r s m e y e r W. Z.: Pflanzenernähr., Düng.,

Bodenkunde, 88, 1960, 203.[36] S o w d e n F. J.: Soil Sei., 80, 1955, 180.[37] S t e e 1 i n к C.: Geochim. et Cosmochim. Acta, 28, 1964, 1615.[38] S t e v e n s o n F. J.: Soil Sei. Soc. Am. Proc., 20, 1956, 204.[39] S t e v e n s o n F. J., C h e n g C. N.: Geochim. et Cosmochim. Acta, 34, 1970, 77.[40] S t e v e n s o n F. J., K i d d n e r G., T i l o S. N.: Soil Sei Soc. Amer. Proc.,

31, 1967, 71.[41] S w a b y R. J., L a d d N. J.: Trans. Int. Soil Conf., New Zealand, Comissions

IV and V, 1962.[42] S z m u k A. A.: Trudy Kubańsk. S-Ch. Inst., t. 1, wyd. 2, 1924.[43] T r u s o w A. G.: Ż. Opytnoj Agronomii, 17, 1916.[44] Т у l i n A. T.: Soil Sei., 45, 1938, 342.[45] W a к s m a n S. A.: “Humus”, 2nd ed., The Williams and Wilkins Co., Baltimo ­

re, USA 1938.[46] W i l s o n R. F., T i l l e y J. M. A.: J. Sei. Fd. Agric., 16, 1965, 173.[47] W o j t a s z e k T., C z u c h a j o w s k a Z.: Coll. of Papers pres, to the Intern.

Congress of Horticulture, Israel 1970.[48] Y o u n g J. L., M o r t e n s e n J. L.: Res. Circ. 61, Ohio Agr. Expt. Sta., 1958.

L. CZUCHAJOWSKI, A. ERNDT

SKŁAD AMINOKWASOWY BIAŁKOWEGO KOMPONENTA KWASÓW HUMINOWYCH Z GLEBY

Katedra Chemii Ogólnej WSR w K rakowie

S t r e s z c z e n i e

Z gleby brunatnej właściwej, wytworzonej z lessu (Prusy) oraz z gleby torfowej, wytworzonej z torfu niskiego na ile mioceńskim (Szarów), otrzymano w wyniku ekstrakcji 0,2n NaOH w temperaturze pokojowej oraz wytrącania kwasem solnym — kwasy huminowe I (Prusy) i II (Szarów), różniące się znacznie zawartością azotu: I — 2,77%, II — 4,00°/o. Kwasy te poddano hydrolizie w warunkach beztlenowych przy udziale 6n HC1 i w otrzymanych hydrolizatach oznaczono zawartość poszczególnych aminokwasów; analizę prowadzono na automatycznym analizatorze Beckmann-Spinco 120B. Ponieważ hydroliza nie przeprowadziła całej ilości kwasów huminowych do

Amino acids composition... 89

roztworu, pozostały osad poddano powtórnej hydrolizie, po czym we wtórnych hy­drolizatach oznaczono zawartość aminokwasów. Wyniki analizy elementarnej w yj­ściowych kwasów huminowych oraz nie zhydrolizowanych pozostałości po pierwotnej i wtórnej hydrolizie, a także ubytek zawartości azotu podano w tab. 1, zaś wyniki analizy ilościowej aminokwasów uwolnionych z części białkowej kwasów humino­wych w wyniku hydrolizy pierwotnej i wtórnej przedstawiono w tab. 2; przytoczone w niej wartości liczbowe podają, jaki procent azotu ogólnego, znajdującego się we wszystkich oznaczonych aminokwasach, przypada na każdy aminokwas. W tab. 3 porównano wartości średnie poszczególnych oznaczeń z tab. 2 ze składem aminokwa- sowym białek roślinnych [46, 47] oraz zhydrolizowanych gleb [36, 38, 48].

Pierwsze hydrolizaty kwasów huminowych obu typów — Ia i IIa — wykazały podobny skład aminokwasowy; w większym stopniu różniły się jedynie zawartością ALA. Różnice składu aminokwasowego między IA i I I a mieściły się w granicach róż­nic znalezionych np. dla frakcji cytoplazmatycznej i chloroplastowej białka liści Phaseolus vulgaris [47]. Drugie hydrolizaty — Ib i Ив wykazały nieco większe różnice składu aminokwasowego, przekraczały one 1,5%> zawartości sumarycznego azotu ami­nokwasowego w przypadku: LYS, HIS, ARG, ASP, GLU, GLY, ALA i VAL. Jednakże, co jest charakterystyczne, prawidłowość wzrostu lub spadku zawartości aminokwa­sów przy przejściu od pierwotnych do wtórnych hydrolizatów okazała się dla kwa­sów huminowych obu gleb taka sama: IIa-^ IIb , I a ^ I b . Wzrastała zawartość LYS odpowiednio o ok. 8 i 6°/o ogólnego azotu aminokwasowego, przypadającego na ten aminokwas, HIS o 7 i 3%, ARG o 9 i ll°/o, VAL o 4 i 2%> oraz ILEU o 4 i 3°/o; zmniej­szyła się natomiast zawartość ASP o 4 i 7°/o i ALA o 4 i 4°/o. Charakterystyczne jest to, że skład pierwszych hydrolizatów obu kwasów huminowych Ia i Ha znacznie bardziej przypomina skład białka roślinnego niż skład wtórnych hydrolizatów I b , H b .

Można pokusić się o sugestię, że w naturalnych kwasach huminowych istnieją dwa typy głównych frakcji białkowych różniących się istotnie składem aminokwa- sowym, albo też występują białka, które podczas hydrolizy wykazują dość podobną od­porność wiązań peptydowych, łączących określone aminokwasy, co powoduje, iż biał­ka te ulegają swego rodzaju hydrolizie „frakcjonowanej” do dwóch frakcji, różnią­cych się od siebie składem aminokwasowym.

JI. ЧУХАЙОВСКИ, А. ЭРНДТ

АМИНОКИСЛОТНЫЙ СОСТАВ БЕЛКОВОГО КОМПОНЕНТА ГУМИНОВЫХКИСЛОТ п о ч в ы

К аф едра Общей Химии, Вы сш ая С ельскохозяйственная Ш кола в К ракове

Р е з ю м е

С типичной бурой почвы образованной из лесса (м. Прусы) и с торфяной почвы образованной из низинного торфа залегающего на миоценовом иле (м. Ша­ров) были получены — в результате экстрагирования 0,2 н NaOH при комнатной температуре и осаждения соляной кислотой — гуминовые кислоты I (Прусы; и II (Шаров) заметно различающиеся по содержанию азота: I — 2,77, II — 4,00%. Эти кислоты были затем подвергнуты гидролизу в анаэробных условиях под воздействием 6 н НС1. В полученных гидролизатах определялось содержание от­

90 L. Czuchajowski, A. Erndt

дельных аминокислот; анализы выполняли на автоматическом анализаторе Beckmann-Spinco 120 В. В виду того, что во время гидролиза не все аминокислоты переходили в раствор, остаток подвергали повторному гидролизу и определению аминокислот во вторичных гидролизатах. Результаты элементарного анализа исходных гуминовых кислот и негидролизуемого остатка после первого и второго гидролиза, а также убыль в содержании азота, поданы в таб. 1; результаты коли­чественного анализа аминокислот освобожденных из белковой части аминовых кислот в последствии первого и второго гидролиза показаны в таб. 2; помещенные в ней цифровые данные показывают какой процент общего азота, находящегося во всех определяемых аминокислотах, приходится на каждую из аминокислот. В таб. 3 сравниваются средние значения отдельных определений из таб. 2 с ами­нокислотным составом растительных белков (46, 47) и гидролизованных почв (36,

38, 48).

Первые гидролизаты гуминовых кислот, Iа и IIа, обладали подобным амино­кислотным составом; более заметно они различались единственно по содержанию ALA. Различия в аминокислотном составе между 1л и IIа находились в пределах разниц выявляемых например для цитоплазматической и хлоропластной фрак­ции белка листьев Phaseolus vulgaris [47]. Вторые гидролизаты — I/з и Ilß пока­зывали немного большие разницы аминокислотного состава, которые превышали 1,5% от суммарного содержания азота аминокислот в случае: l y s , h i s , a r g , a s p , g l u , GLY, ALA и v a l . Однако, что является характерным, закономерности в повы­шении либо понижении содержания аминокислот при переходе от первичных к вторичным гидролизатам были для гуминовых кислот почв аналогичны: Н А — I I В, I А — Iß- Повышалось содержание l y s на около 8% а общего аминокислотного азота, приходящегося на эту аминокислоту, на 6%; h i s — соответственно на 7%

и 3%; a r g — на 9% и 11%; v a l — на 4% и 2°/о, а также i l e u на 4°/о и 3°/о; пони­зилось ж е содержание a s p на 4% и 7%, a a l a на 4°/о и 4°/о. Характерно, что состав первых гидролизатов обеих гуминовых кислот, Iа и Пл, был заметно более бли­зок составу растительного белка, чем состав вторичных гидролизатов Iß и Пл.

Авторы склонны полагать, что в естественных гуминовых кислотах имеются либо — два типа главных белковых фракций существенно различающихся в отношении аминокислотного состава, либо —■ выступают в них белки, которые во время гидролиза проявляют сходную силу пептидных связей соединяющих данные аминокислоты, что приводит к своего рода „фракционированному” гидро­лизу этих белков до двух фракций, отличающихся друг от друга аминокилсотным составом.

Wpłynęło do PTG w lipcu 1970 r.Dr Leszek Czuchajowski Zakład Chemii Ogólnej U niw ersytetu Jagiellońskiego Kraków