LESZEK CZUCHAJOWSKI, ALEKSANDER ERNDT
AM INO ACIDS CO M POSITION OF PRO TEIN A CEO U S COM PONENT OF SO IL HUM IC ACIDS
Department of General Chemistry, U niversity College of Agriculture, Kraków
HUMIC ACIDS STRUCTURE AND THEIR NITROGENOUS COMPONENTS
T he organic m a tte r of th e soil is a com plex of sub stances th e com posi tio n of w hich is d e term in ed in p a rt by th e p la n t and an im al residues added to th e soil, and to a g re a te r ex ten t, by th e tra n sfo rm atio n s of th ese su b s tra te s th ro u g h physical, chem ical and biological m eans. Soil organic m a t te r contains m ost, if not all, of th e n a tu ra lly occurring organic com pounds. In additio n to th e in te rm e d ia ry p roducts of organic m a tte r break d o w n and secondary sy n th e tic pro d ucts of m icrobial action, th e re exists in th e soil an accum u lation of th e m ore re sista n t p ro du cts of decom position — th e soil hum us.
The n ativ e organic fractio n of soils is m ade up of a h eterogen ou s m ix tu re of polym erized arom atic m olecules, polysaccharides, bound am ino acids, uronic acid polym ers, and various organic phosphorus com pounds [27]. By definition, hum us is a com plex m ix tu re of am orphic and colloidal substances arising from m odified p la n t m ate ria ls an d synthesized m icro bial tissue.
T he c h a ra c te risa tio n of hu m u s is fa r from com plete, and concepts con cerned w ith its fo rm ativ e processes are, to a larg e ex ten t, speculative. E arly th eo ries p o stu la te d th a t lignin com plexed w ith p ro te in of m icrobial origin provided th e m ain source of hum ic substances in soil. H um ic acids w ere found to yield d eg rad atio n p ro d ucts id entical w ith those obtained from lignin. T he sim ila rity b etw e en th e aro m atic s tru c tu re of lig nin and th a t found in hum ic acids, and th e re la tiv e resistan ce of lignin to enzym a tic break d o w n m ay be said to be in good a g reem en t w ith th e lign in th eo ry of h um u s fo rm atio n [45].
M ore rec e n t studies, how ever, suggest th a t m an y com pounds of non- lignin origin m ay also be a source of aro m atic “s tru c tu ra l c lu ste rs” . Mi cro-organism s a re able to give rise to com pounds of an aro m atic or h e te ro - arom atic n a tu re th ro u g h th e conversion of m an y diverse organic com pounds including c a rb o h y d rates [34]. Such aro m atic stru c tu re s can be oxi dized and condensed w ith am ino acids or p ep tides w ith th e fo rm atio n of substances sim ilar to those found in th e soil hum ic acid fraction. It app ears at th e p re se n t tim e th a t th e “n u clei” of hum ic acids arise both from a lte red lignin and ta n n in com pounds and th ro u g h th e sy n th esis of arom atic com pounds by m icro-organism s. It seem s p ro b able th a t m an y p ro du cts of synthesis and m icrobial m etabolism such as am ino acids, peptides, am i- nosugars, nucleoproteins, p u rin e and p y rim id in e bases, uronic acids, orga nic acids, alcohols, etc., p a rtic ip a te in th e fo rm atio n of th e fu n ctio n al groups of hum us.
The ch em istry of soil organic m a tte r is b e st stu d ied in th e absence of inorganic m a trix of soil and th e re fo re th e organic m a tte r m u st firs t be dissolved by an aqueous or nonaqueous e x tra c ta n t. A t p re se n t available ex tra ctio n pro cedu res are ra th e r em pirical, and th e com plete e x tra ctio n seem s to be a n im possibility. On th e o th er hand, m ost e x tra ctio n proced ures p ro b ab ly p roduce artifa c ts, organic m a tte r e x tra c te d from th e soil m ay be v e ry d iffe re n t from th a t w hich ex ists in th e soil. O xidation, p o ly m eri zation and photochem ical reactio n s can change to some e x te n t th e p a re n t m ate ria l d u rin g ex tractio n .
E x tra c tio n is m ade d ifficu lt by th e ad so rptio n of organic m a tte r on m in e ral co n stitu en ts of th e soil. D ue to th e ir g re a t su rface a rea and p re pond eran ce of rea c tiv e ad so rp tio n sites, clay m in erals low er th e e x tra ctio n efficiency. It has been show n [21] th a t p o lar com pounds and non-ionic com pounds a re adsorbed on clay, p a rtic u la rly on th e basal su rfaces of ex panding clays. A liphatic m olecules a re o rien ted w ith th e plane of th e ir chains bo th p a ra lle l and p e rp e n d icu la r to th e clay surface. Some organic m olecules a re held to th e clay su rface th ro u g h С—H " '0 bonds [3, 12, 29], a lth o u g h an y ad so rb ate capable of supplying electrons to th e incom plete p o rbitals of adsorbed or lattic e alu m in iu m could be adsorbed as a carbon ium ion [25]. D etailed stu d y has show n [11] th a t exposed la ttic e a lu m in ium or iro n are involved in th e ad so rption of c a rb o h y d rates on clay. O rganic cations and polycations, such as proteins, below th e isoelectric poin t a re adsorbed on clay b y a catio n -ex ch ang e m echanism . T hese cou- lom bic forces a re p ro bab ly su p p lem en ted b y van d e r W aals forces and С—H""0 bonds b etw een th e organic m olecule and th e clay m in e ral s u r face. T he use of a basic e x tra c ta n t rem ove adsorbed am photeric com pounds. A n increase in b asicity re n d e rs th e organic m a tte r electron eg ativ e and p rev e n ts ex tensiv e ad so rptio n by coulom bic rep u lsio n forces [32].
In ad dition to th e q u a n tity of organic soil m a tte r h e ld to th e clay s u r face, th e re a re indications th a t significant am o unts of organic m olecules in soil and sed im ents occur in in tim a te association w ith clay [40]. It has b een suggested th a t am ino acids and p ep tid es a re adsorbed on in te rla - m ella r surfaces of clay m inerals. R esu lt of X -ra y d iffra c tio n stu dies have show n th a t th e re is an a p p a re n t “c o n tractio n ” of th e thickness of am ino acids and p eptides by ad so rp tio n on in te rla m e lla r positions of m ontm o- rillonite, indicated th a t th e adsorbed m olecules a re “k e y e d ” into hexag on al holes [19]. A m ino acids or p eptides boun d to clay in th is m a n n e r w ould be p a rtic u la rly d ifficu lt to rem ove by chem ical or biological agents. T he ad so rptio n of am ino acids b y clay p rovides a p a rtia l ex p lan atio n for th e ir a b ility to su rv iv e in sed im ents and se d im e n ta ry rocks over geologically long periods of tim e [39].
A n o th e r facto r w hich affects th e so lu bility of organic m a tte r is th e fo r m atio n of salts and gels w ith m eta l ions. T he org an o -m in eral colloids in soil a re a com plex m ix tu re of floccules and gels [44], th e s ta b ility of w hich depends on th e pH and o x id atio n -red u ctio n sta tu s of th e soil [1]. These organom ineral gels a re tra n sfo rm ed into w a te r-so lu b le h yd roxy-co m plex es by p a rtia l hydrolysis in a slig h tly alkalin e m edium . In a stro n g ly alk alin e m edium th e y a re com pletely h y dro ly sed [26].
T he rem oval of th e organic p hase b y solvent e x tra ctio n of th e soil gives a larg e n u m b er of re la tiv e ly sim ple com pounds of kn ow n s tru c tu re s such as carb o h y d rates, carboxylic acids, esters, am ino-acids, glycosides an d o r ganic bases. T he re m a in d e r of th e soil organic m a tte r consists of a m ix tu re of am orphous b ro w n or black p ro d u cts re fe rre d to as “h u m u s” , and p a r tia lly decom posed p la n t and anim al residues. “H u m us” has b een se p ara te d into fractio ns d ifferin g in so lu bility in w a te r, alkalis, acids an d organic solvents, b u t in view of th e h ete ro g en e ity of th e fractions d iffe re n t te rm i nologies have b een used b y d iffe re n t au tho rs. In th e course of th e p re se n t w ork, th e te rm “hum ic acids” is used for th a t p a rt of h um us w hich is soluble in 0.5 N sodium h y d ro x id e and p rec ip ita te d by acidification of th e alk alin e e x tra ct. Such a d efin itio n has b een w idely used, alth o u g h O d e n [33] rem o ved th e alcohol-soluble p a rt of th e p re c ip ita te as “h y m ato m e- lanic acids” ; it is, how ever, d o u b tfu l w h e th e r such a sub-division is desi rab le [2]. T he te rm “fulvic acids” re la tin g to th e po rtio n of “h u m u s” w hich rem ain s in th e aqueous liq u o r a fte r th e acidification of th e alk aline e x tra ct, is still re ta in e d by m an y w o rk ers alth o u g h th is fractio n includes polysaccharides, peptides, am ino acids an d re la tiv e ly sim ple phenols as w ell as com pounds of un k now n stru c tu re .
T he com position of hum ic acids is d e te rm in e d by th e source and isola tio n technique. E le m e n ta ry an aly ses of p ro d u cts isolated fro m vario us soils have given values rang ing for С 49-62% , H 3-6 % . N 0.4-5.0% , and
m olecular w eig hts from 1000 to 300,000 a re re p o rte d [2]. D eg rad atio n of
hum ic acids involving fusion w ith alk ali [22], oxidation w ith n itric acid
[42], p e rm a n g an a te [30], alk alin e n itro b en zen e [31], or copper oxide [18] gave sm all yields of phenols such as catechol, resorcinol, and p h lo ro glu - cinol, aldehydes including p -hy d ro x y -b en zald eh y d e, vanillin, sy rin gald e- h yde and acids such as p-hydroxybenzoic, vanillic, protocatechuic, v e ra t- ric and isohem ipinic acids.
R ecent stu d y [4, 24] have show n th a t som e of th e phenols, based upon resorcinol or phloroglucinol stru c tu re s w ere a ttrib u te d to flavonoid p re cursors, o th ers based upon catechol ty p es w ere reg a rd e d as lig n in -d eriv ed units. It w as th e n concluded th a t besides lignin, flavonoids and possibly o th er n a tu ra lly occurring polyphenols m ay be involved in th e fo rm atio n of hum ic acids, as suggested ea rlie r by T r u s o v [43].
In fra re d ab so rptio n bands of hum ic acids in th e region of 1600 cm -1,
1510 cm-1 and 840 cm“ 1 are co rrelated w ith th e presence of arom atic rings
[35]. In p a rticu la r, rin g s of th is ty p e have been po stu lated in a q u a n tu m ch em istry in te rp re ta tio n of in fra re d abso rp tio n changes w hich ap p eared by p yrolysis of hum ic acids and of m odel arom atic com pounds having OH groups [6, 10]. M ore recen t ex p erim en ts [5] on th e d eg rad atio n of hum ic acids using zinc d u st distillatio n a t 500°C in a stre a m of hydrog en have proved th e presence of arom atic nuclei in th e hum ic acid m acrom olecule. A n th racen e and 2,3-benzofluorene w ere isolated in cry stallin e p u re form from th e d estillate. A com bination of UV sp ectro m etry , v ap ou r phase chro m ato g rap h y and m ass sp e ctro m e try provided convincing evidence of th e presen ce of nap h th alen e, 1- an d 2-m e th y ln a p h th a le n es and a w ide ran g e of o th er aro m atic h ydrocarbons in th e d istillate, including pyrene,
perylene, 1,2-benzopyrene, and coronene, to g eth e r w ith hom ologues, and
also the presence of hom ologous acridines and 1,2- or 3,4-benzoacridines. In a subseq u en t e x p e rim e n t oxidation of hum ic acids w ith p e rm a n g an a te follow ed by d ecarb o x y latio n in quinoline and C u S 04 gave an th raq u in o n e, m eth y la n th ra q u in o n e s, 2-m e th y ln a p h th a le n e, fluorenone, xantho ne, m e- th y lx a n th o n e s and th e h y drocarbons C13H 12, C14H10 and Ci5H 14. E ven though th e p rod u cts from th e zinc d u st d istillatio n m ay arise from secondary r e actions th is is im probable in th e case of pro d u cts from th e p e rm a n g an a te oxidation. C onsequently, it is believed th a t th e existence of a po lyn u clear arom atic s tru c tu re in hum ic acids has b een established.
E lectron p aram ag n etic resonance spectra have show n [37] th a t soil
hum ic acids contain stable organic free rad icals of th e ord er of 1018 spin/g.
These rad icals a re ex ceptionally stab le w ith resp ect to tim e and chem ical attack . T he sam ples have show n no d etectab le decrease in spin concen tra tio n s over a period of th re e y ears. E x tensive acid hy drolysis of hum ic acids only increased th e free rad ical co n ten t in th e hyd ro ly sate; th is sam e
b eh av io u r w as enco u n tered w ith high te m p e ra tu re oxidation, w h e re as th e red u ctio n of hum ic acids w ith sodium m eta l and lith iu m alu m in iu m h y dride caused only a sm all decrease in spin co ncentration.
It w ould a p p e ar th a t fre e radical species are an in te g ra l p a rt of th e n a tu ra l hum ic acids m acrom olecules and th e rad ical s tru c tu re is e ith e r shielded by th e polym eric n etw o rk or stabilized by delocalization of th e u n p a ire d electrons over a su itab le arom atic system .
As a significant increase in spin co n cen tratio n has been observed for th e sam ples w h en th e y w ere co n v erted to th e sodium salts, it is assum ed th a t hum ic acids con tain sem iquinone radicals co existent w ith q u in h y - drone m oieties, pro bab ly d eriv ed from ortho or p ara benzoquinones.
It is ch aracteristic th a t our previous stu d y [8, 9] on th e origin of th e absorptio n b and n e a r 1600 cm-1 in th e in fra re d sp ectra of hum ic acids have p o stu lated th e presence of h y d ro x y p h en y l-q u in o n es and of some sim ple condensed h y d ro x y ary lq u in o n es “in form of sem iquinones and h y d ro - quinone-q u ino ne c h a rg e -tra n sfe r com plexes, as th e s tru c tu ra l u n its in th e hum ic acids m acrom olecules.
T he g en eral s tru c tu re (I) has re c e n tly been advanced [5] in w hich h u mic acid is a com plex consisting of a polycyclic arom atic “core” a tta ch e d to polysaccharides, proteins, rela tiv e ly sim ple phenols and m etals.
Carbohydrates --- Polypeptides
Polycyclic x aromatic core
Phenolic acids --- M etals (I)
Most of th e carb o h y d rates a re p rob ably p re se n t as polysaccharides. F o r s y t h [16] show ed th a t boiling w a te r rem oved from hum ic acids de
rived from a v a rie ty of sources, about 20% as a polysaccharide m ix tu re
yielding on acid hy drolysis glucuronic acid, galactose, glucose, m annose, arabinose, xylose and ribose. R are 4-O -m eth y l-D -g alactose and 2-O -m e- th y l-L -rh a m n o se have b een isolated [13] from a p e a t polysaccharide.
T he rem o val of carb o h y d rates and phenolic acids b y m eans of cold or hot w a te r is not necessarily evidence in fav o u r of physical adsorption; rea d ily h y d roly sab le depsidic, e ste r and glycosidic linkages a re fre q u e n tly en co un tered in o th er fields, e. g. th e gallo- and ellagitam ins [20], an d th e hyd rolysis of hum ic acids w ould p ro b ab ly be fac ilita te d by th e p resence of carboxy l or o th er acidic groups in th e m olecule. T h ere is no d o ubt th a t polysaccharides of th e hum ic acid fra c tio n of th e soil p lay a n im p o rta n t
role in th e cation -ex ch ange capacity, carbon m etabolism , and com plexing of m etals in soil.
T he m ode of a tta c h m e n t of th e protein aceous co n stitu e n ts is still u n certain . H ow ever, m an y au th o rs [28, 41] considered th a t th e sta b ility to w ard s chem ical and m icrobial a tta c k of th e p ep tides w as an in dicatio n th a t th e y a re an in te g ra l p a rt of th e hum ic acid m olecule. T he sta b ility m ay be caused b y chem ical com bination of th e p ep tides w ith quinone groups by rea c tio n sim ilar to th ose fre q u e n tly p o stu la te d in e a rlie r th eo ries of tanning.
S tud ies on m odel substances (II, III, IV) have show n [5, 14] О
R y II R = —NH • CH2 • CO • OC2H5
Y ) III R = — CH3
II \ n h • CH2 • C O • OC2H 5 IV r = _ o c h 3
о
th a t all th re e com pounds lost glycine on h y drolysis w ith 6 N HC1 ju st as
th e n itro g e n w as rem oved fro m hum ic acids by hydrolysis. S im u lta neously, in all th re e cases black am orphous substances w e re produced, p resu m ab ly by f u rth e r condensation of th e h y d ro xy -p -benzo qu ino nes resu ltin g from th e rem o val of th e glycine. H ow ever, all th re e com pounds in v estig a ted are u n lik e ly to lose th e ir n itro g en by tre a tm e n t w ith w a te r, a n d th e rem oval of a sm all am o u nt of p e p tid e -lik e substances by e x tra c tion w ith cold or hot w a te r suggests e ith e r p hysical a tta c h m e n t or a lte r natively, a tta c h m e n t by h y d ro g en bonding b etw e en th e im ide groups of th e pep tid e and phenolic groups (stru c tu re V or VI).
HO -> H—N— C-i i и ^ R O V xR fH—N< -Q / > \ r <-o^ VI
Som e n itro g en in hum ic acids is stable against hydrolysis w ith 6 N HC1
and un affected b y m éth y la tio n and acéty latio n [15, 23] and m ay be p re s e n t in heterocyclic form .
T he p re se n t stu d y w as u n d e rta k e n to d e te rm in e q u a n tita tiv e ly th e am ino acid com position of th e p ep tid e and p ro te in content of soil hum ic acids, its c h a ra c te ristic response to th e conditions of hydrolysis, and to in v estig ate th e re la tio n to th e am ino acid com position of th e soil and g reen p la n t species.
EXPERIMENTAL
As a re s u lt of co llaboration w ith th e D e p a rtm en t of Soil Science, U n iversity College of A g ricu ltu re, Cracow, it has b een possible to exam ine hum ic acids e x tra c te d from th e follow ing soils.
Soil I; source: A g ricu ltu ra l E x p erim en t S tatio n (RZD) P ru sy , field B, profile located ca. 300 m from th e road. P a re n t rock: loess; p la n t cover: arab le field p rep a re d for e x p e rim e n tal w ork, sam ple from horizon A x (В ) 25-30 cm, b ro w n -g re y fine sand w ith hu m u s streak s, slightly m oist, cru m b s tru c tu re , loose, no roots, pH 7.0; soil type: p ro p er brow n soil, soil fam ily: form ed on loess, te x tu ra l group: fine sand;
Soil II; source: p e a t m eadow , Szarów. P a re n t rock: low m oor fen te n d ing to tra n sitio n m oor, on M iocenian clay; p la n t cover: R an un culu s sp.,
T araxacum p a lu stre , Plantago lanceolata, B eilis p e ren n is, Poa a nnua,
sam ple T 2, 51-126 cm, brow n p e a t slig h tly decom posed, w et; soil type: p eat soil, soil fam ily: form ed on low m oor fen w ith u n d erly in g M iocenian clay; p e a t species: A ldersedge peat.
ISO LATION OF HUMIC ACIDS
A ccording to th e sta n d a rd p ro ced u re [2], th e a ir-d rie d soil sam ple w as d e fa tte d w ith a m ix tu re of equal volum es of e th y l alcohol and benzene, and d rie d a t 90°C. T he d e fa tte d soil (20 g) and 250 cc of 0.2 N NaOH, a fte r standin g a t room te m p e ra tu re for 18 h rs w ith occasional shaking, w ere centrifuged. T he s u p e rn a ta n t liquors w e re filte re d th ro u g h a sin te re d glass fu n n el (No. 4) an d th e filtra te w as acidified w ith 0.2 N HC1 to pH 1. For collection of th e hum ic acids a m odified F o r s y t h a nd F r a s e r ’s tech n iq u e [17] w as used, in w hich th e acidified m ix tu re w as cen trifu ged , th e liq u o r d ecan ted and th e m oist p rec ip ita te w as frozen to — 50°C for 12 hrs. Then, it w as allow ed to th aw an d th e solid collected on a sin tered glass fu n n el (No. 4), w ash ed u n til free from chloride, and d rie d a t 90°C.
T he y ield of hum ic acids e x tra c te d from th e soil I w as 0.2*Vo, and from th e soil II — 26’°/o, respectively . T he re su lts of elem en tal an alyses 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 : T he sam ple of 0.2 g of hum ic acids and 20 cc
of 6 N HC1 w e re placed in a glass tu b e w hich w as th e n sealed u n d e r
nitrogen. T he h y drolysis w as carried out by h eatin g th e tu b e co n ten t in an oven a t 110°C for 20 hr. Then, th e tu b e w as opened, th e liq u o r filte re d th ro u g h a sin te re d glass fu n n el (No. 4) and th e solid w ashed w ith th re e portions of 25 cc dist. w ater. T he com bined filtra te w as ev ap o rated to
T a b e l a 1
E lem entary a n a ly s is o f s o i l humic a c id s and o f th e r e s id u e s a f t e r t h e i r h y d r o ly s is E lem entary с c a p o s it io n /% / D ecrease o f n it r o g e n i n % o f i t s c o n te n t in : No. M a te ria l С H N pa ren t humic a c id s r e s id u e a f t e r f i r s t hydro l y s i s
1 Humic a c id s d e r iv e d from th e brown l o e s s ,
P ru sy , I 5 2 ,5 1 4 ,8 4 2 ,7 7 2 R esid ue a f t e r f i r s t h y d r o ly s is 1 ,5 3 1 ,3 6 > 4 % 3 o f I R esid ue a f t e r second h y d r o ly s is 4 5 ,3 1 3 ,5 8 > 11%
4 Humic a c id s d e r iv e d fr o n th e peCst meadov/,
S żarów, I I 5 3 ,4 9 5 ,3 4 4 ,0 5 R esidue a f t e r f i r s t ' h y d r o ly s is o f I I R esid ue a f t e r second h y d r o ly s is 5 7 ,9 7 5 9 ,4 2 4 ,7 6 4 ,3 9 o in > 4 S £ 6 1 ,2 3 >3C?& T a b e l a 2
Amino a c id c o m p o sitio n o f p r o te in a c e o u s component o f s o i l humic a c id s ; th e d a ta e x p r e s s a p e r ce n ta g e n it r o g e n c o n tr ib u tio n o f each amino a c id i n th e t o t a l
n it r o g e n c o n te n t o f a l l amino a c id s rec o v e r ed
Humic a c id s from th e brown l o e s s s o i l , Humic a c id s from th e p ea t meadow s o i l ,
Amino P r u sy , / I / Szarów, / I I / a c id F i r s t h y d r o ly z a te s , 4 Second h y d r o ly z a te s , ZB F i r s t h y d r o ly z a te s , h y d r o ly z a te s ,Gecond IXb LYS 9 ,9 6 ,8 5 ,2 1 5 ,8 2 4 ,0 8 ,3 1 0 ,4 9 ,4 2 2 ,0 1 2 ,0 HIS 4 ,8 2 ,3 1 .3 6 ,2 X 4 ,0 3 ,0 3 ,6 1 0 ,5 X ARG 8 ,8 4 ,0 3 ,5 1 6 ,0 X 6 ,6 5 ,4 3 ,5 1 3 ,9 X ASP 1 3 ,4 1 2 ,2 1 4 ,3 6 ,2 6 ,6 1 3 ,2 9 ,9 1 4 ,2 7 ,7 8 ,3 THR 4 ,0 4 ,8 6 ,4 2 ,6 3 ,4 5 ,2 4 ,5 6 ,0 3 ,0 3 ,0 SER 3 , 4 4 ,7 5 ,7 2 ,6 4 ,0 4 ,1 4 , 4 4 ,9 3 ,1 4 ,3 GLU 9 ,3 1 0 ,7 9 ,4 6 ,1 8 ,3 8 ,9 9 ,0 1 0 ,6 8 ,0 1 0 ,2 PRO 6 ,4 9 ,5 8 ,1 4 ,7 7 ,1 5 ,0 3 ,1 8 ,0 4 ,5 6 ,9 GLY 8 ,3 1 0 ,1 1 3 ,4 6 ,2 8 ,3 1 0 ,1 1 1 ,4 1 2 ,2 9 ,2 1 1 ,1 ALA 8 ,3 1 0 ,1 9 ,6 4 , 4 6 ,1 1 2 ,0 1 0 ,4 1 1 ,5 5 ,9 8 ,0 VAL 6 ,7 6 ,2 6 ,7 9 ,3 8 ,6 6 ,8 6 ,2 8 ,6 1 1 ,3 1 0 ,6 MET 0 ,8 0 ,4 0 ,4 0 ,2 0 ,9 0 ,4 0 ,3 0 ,5 0 ,0 5 tr a c e s I LEU 3 ,7 3 ,5 3 ,8 6 ,2 7 ,0 3 ,7 3 ,8 5 ,3 7 ,5 8 ,5 LEU 6 ,7 6 ,8 6 ,7 6 ,4 8 ,2 6 ,3 6 ,0 8 ,0 8 ,1 9 ,9 TYR 1 ,7 1 ,5 1 ,4 2 ,6 2 ,1 1 ,8 1 ,1 1 ,1 2 ,0 2 ,1 PHEN 3 ,7 5 ,4 3 ,9 4 ,6 4 ,8 4 ,0 5 ,6 3 ,8 4 ,9 5 ,0 Uniden t i f i e d Long c o l . , b u f f e r I I :
ca. 53 m in ., ca 75 min. a b sen t
Long c o l . , b u ffe r I I :
dryness a t 40-45°C an d used for d e te rm in a tio n of am ino acids. T he solid collected on th e fu n n el w as d ried in vacu um to co n stan t w eight, analyzed for C, H, N co n ten t and reta in e d for n e x t hydrolysis.
S e c o n d h y d r o l y s i s : T he d ried sam ple (0.2 g) of th e solid resid u e
a fte r firs t hyd ro ly sis and 20 cc of 6 N HC1 w ere placed in a glass tube,
sealed u n d e r n itro g en and tre a te d as d escribed above. T he evap o rated f iltra te w as used for th e d e te rm in a tio n of am ino acids. T he rem ain in g resid u e w as an aly sed for C, H, N, content.
B oth hy dro lysis p ro ced u res w e re c a rrie d out on rep lica te sam ples and all d e te rm in a tio n s re p e a te d tw ice.
T he re su lts in T able 3 show th e m ean valu es of tw o or th re e se p ara te estim ations; th ese a re p rese n ted in T able 2.
AM INO ACIDS A N A L Y SIS
T he analyses w e re c a rrie d out on th e B eckm ann-Spinco 120B am ino acids au tom atic an alyser, fitte d w ith tw o colum ns. T he longer one, 50 cm long an d 0.9 cm in d iam eter, filled w ith C ustom S p herical R esin T ype
AA-15 (m inim al 8'°/o crosslinked sulp h o n ated sty re n e copolym er), w as
elu ted in a s ta n d a rd w ay w ith 0.2 M citric b u ffe r a t pH 3.28. In about 150 m in u tes b u ffe r I w as replaced by citric b u ffe r II, pH 4.25. T he sh o rt colum n, 10 cm long and of th e sam e d iam eter, filled w ith AA-27 resin, w as elu ted w ith citric b u ffe r pH 5.28. T he in v estig ated sam ples w e re in tro duced on colum ns a fte r dissolving th em in 0.1-2 cc of th e b u ffer. T he co n cen tratio n ord er of th e solution d eriv ed from th e in te n sity of ch ro m a togram s.
T he rep ro d u c ib ility of m axim a positions of th e calib ratio n m ix tu re com ponents w as p e rfe c t as long as th e sam e solution of n in h y d rin re a g e n t and of th e b u ffers w ere used th ro u g h o u t th e analysis. H ow ever, in analyses c arried out w ith new ly p rep a re d portions of n in h y d rin rea g e n t and of th e b uffers, th e positions for in d iv id u al am ino acids varied: for th e com pounds ap p earin g w ith in th e first 70 m in u tes (long colum n separation) th e change w as w ith in th e ran g e of 2 m in., fo r those ap p earin g la te r w ith in th e ran g e of 4 m in. By th e sh o rt colum n se p ara tio n th e differen ces w ere in th e
ran g e of 2 m in., except for ARG w hich w as rev ealed at in te rv a ls of
4-5 min.
E lutio n tim e fo r th e am ino acids w as as follows; sh o rt colum n: LYS
(ORN) 27, H IS 37, ARG 68; long colum n, I b u ffer: A S P 45, TH R 53, SER 56,
GLU 64, PRO 74, GLY 88, ALA 95, ABUT 110, VAL 128; II b u ffer:
ILEU 8-12, LEU 16, TYR 34, PH E 41 m inutes. Q u a n tita tiv e calculations w ere m ade by th e sta n d a rd m ethods using th e calib ratio n curves as referen ce.
Amino a c id com position o f p r o te in a c e o u s component o f s o i l humic a c id s ; mean v a lu e s o f n itr o g e n c o n tr ib u tio n from Tab. 2 , compared w ith th e v a lu e s g iv e n f o r s o i l and p la n t p r o te in h y d r o ly z a te s ,
/ 3 6 , 3 8 ,4 8 / and / 4 6 , 4 7 / Amino a c id Humic a c id s S o i l s from L eaves o f P h aseо lu s v u l g a r i s , p r o te in f r a c t i o n s / 4 7 / Lucerne / 4 6 / Grass p r o te in s / 4 6 / P rusy / I / Szarów / I I / f i r s t h y d ro l. 4 second h y d r o l. *B f i r s t h y d ro l. П А second h y d ro l. K B Lacombe / 3 6 / Flanagan, s i l t loam, / 3 8 / H o y t v i l l c , c la y loam, / 4 8 / whole p r o t. cy to plasm . p r o t. A В LYS 7 ,3 1 5 ,4 9 ,4 1 7 ,0 1 5 .7 * 1 3 ,4 1 4 ,0 9 ,5 1 0 ,8 9 ,4 9 .4 9 .4 HIS 2 ,8 6 ,2 3 ,5 1 0 ,5 2 ,4 * 1 .7 2 .5 5 ,8 4 ,4 5 ,1 5 .0 4 .7 ARG 5 ,4 1 6 ,0 5 ,2 1 3 ,9 4 ,1 * 4 ,9 2 ,8 1 6 ,0 1 1 .5 1 6 ,6 1 5 .7 1 4 ,4 ASP 1 3 ,3 6 ,4 1 2 ,4 8 ,0 1 3 ,5 6 ,3 1 3 ,6 8 ,4 1 0 ,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 1 0 ,6 7 ,3 1 1 ,2 1 0 ,2 1 3 ,2 1 2 ,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 1 0 ,7 1 2 ,2 6 ,9 7 ,4 7 ,1 7 ,5 8 ,0 VAL 6 ,5 8 ,9 7 ,2 1 1 ,0 6 ,0 6 ,5 6 ,9 5 ,4 6 ,0 5 ,9 5 ,1 6 ,4 MET 0 ,5 0 ,4 0 ,4 t r a c e s not g iv e n 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
* / n o t g iv e n in / 3 6 / , supplem ented by th e authors*, * * / not g iv e n / 3 8 / , supplem ented by th e authors
L . C z u c h a jo w sk i, A . E r n d t
RESULTS AND DISCUSSION
T he am ino acid co n tents of acid hydro ly zed hum ic acids derived from th e b ro w n loess soil (I, P rusy) and from th e p eat m eadow soil (II, Szarów) are show n in T able 2. T he d ata express a p ercen tag e n itro g en co n trib u tio n of each am ino acid in th e to ta l n itro g en content of all am ino acids recovered. W ith in individual am ino acids w hich a re typical con stitu en ts of p ro te in p rep a ra tio n s try p to p h a n w as not estim ated because of its d e stru c tio n u n d e r th e conditions of hy drolysis em ployed. On th e o th er hand, th e re su lts p rese n ted in T able 2 show ing th e tim e of elu tion of am ino acids from the colum n ind icate th e presence of some u n id en tified com p onents giving a positive rea c tio n w ith n in h y d rin . T h e re a re tw o com pounds ap p earin g in ca. 53rd and 75th m in. of elu tio n w ith b u ffe r II from th e long colum n w hich m ay be reg ard ed as glucosam ine and galactose- am ine.
P re se n ta tio n of a n aly tical re su lts included in T able 3 shows clearly th e re la tio n b etw e en am ino acid com position found in hum ic acids an d th a t of p la n t p ro te in p rep aratio n s. It seem s also u seful to point out certain considerations reg a rd in g th e relatio n sh ip b etw e en am ino acid com position d e te rm in e d in th e p re se n t w o rk and th e am ino acid co n ten t of acid h y dro lyzed soils re p o rte d in th e lite ra tu re [32, 36, 38, 48]. T he com parison of th e am ino acid com position in hum ic acids w ith th a t foun d in p ro tein h ydro ly zates fro m th e leaf of P haseolus vulgaris [47] is w o rth y of special note because of a n id e n tity of th e h y drolysis p roced u re and of th e te c h niqu e of am ino acids analysis.
In o rd er to avoid in e q u a lity of sam ples associated w ith heterog en eou s soil m aterial, each hum ic acids sam ple to be hydro lyzed consisted of several hum ic acids po rtion s e x tra c te d e ith e r from th e b row n loess soil (I, P ru sy ) or from th e p e a t soil (II, Szarów).
In T able 1 a re given th e re su lts of elem en tal analyses of hum ic acids b efore hydro lysis and a fte r th e first and th e second tre a tm e n t w ith 6 N hydrochloric acid. It can be seen th a t th e first hydrolysis low ers th e nitro g e n co n ten t in th e resid u e alm ost equally in hum ic acids I (Prusy) and in hum ic acids II (Szarów). T he d ecrease is 45% in I and 48% in II, respectively , even th o u gh th e p a re n t hum ic acids before hydrolysis d iffered m ark e d ly in th e ir n itro g e n co n ten t (2.77% N in I, 4.0% N in II). T he second hydroly sis causes a d iffe re n tia tio n in th e release of n itro g en from th e resid u e a fte r th e firs t action of 6 N HC1; th e v alues a re 11% and 30'% of th e n itro g e n p re se n t before th e second tre a tm e n t.
No larg e d ifferences in am ino acids co n ten t of th e firs t h y d roly zates of hum ic acids I an d II w ere found, although, at th is stage of hydrolysis th e co n ten t of alan in e w as 11.3% (II) and 9.3% (I). D ifferences in am ino acids co n ten t in th ese h y d rolyzates w ere w ith in th e ran ge of am ino acid
d istrib u tio n in p ro te in p rep a ra tio n s of cytoplasm ic an d chloroplast fra c tions rem ov ed from th e leafs of Phaseolus vulgaris [47].
L a rg er differen ces in th e co n ten t of in d iv id ual am ino acids w e re found in th e second h ydro ly zates of I an d II. T he con tents of LYS, HIS, ARG, A SP, GLU, GLY, ALA and VAL ex pressed as a p erc e n ta g e of th e to ta l am ino acid n itro g e n d iffered b y m ore th a n 1.5'%. H ow ever, a c e rta in re g u la rity of v ariatio n s in am ino acid co n ten t in th e firs t and th e second h y dro ly zates of hum ic acids (II an d I) d eriv ed from b o th ty p es of soil is
n o tew o rth y . As th e action of 6 N HC1 p roceeded th e co n ten t of LYS in
th e h y d ro lyzates expressed as a p ercen tag e of to ta l am ino acid n itro g en
increased by ca. 8% (II) and 6% (I), th a t of H IS b y 7% and 3% , ARG
9% and 11%, VAL 4% and 2% , ILEU 4% and 3% , respectively; th e
co n ten t of A S P d ecreased by 4% an d 7% , an d th a t of ALA w as low er by 4% and 4% , respectively . In fact, th ese differen ces w e re associated w ith all analy sed basic am ino acids an d does not re fe r to th e n e u tra l am ino acids having cyclic fra g m en ts and h y d ro x y l groups.
It is of p a rtic u la r in te re s t th a t th e am ino acid com position of th e first hy d ro ly zates of b o th hum ic acids (I an d II) rem inds m uch m ore of th e d istrib u tio n of am ino acids in p la n t p ro te in of th e aboveground p a rt of
Phaseolus vulgaris [47] and of lu cern e and grass [46], th a n it does th e
am ino acid com position of second hydrolyzates. In th e la tte r, only th e contents of ARG, A SP, PRO an d ALA m ay be re la te d to th e p la n t p ro te in com position.
It seem s also in te re stin g to com pare th e re su lts of th e p re se n t w ork concerning th e am ino acid com position of proteinaceous co n stitu en ts of hum ic acids w ith those of acid hy d ro lyzed soils re p o rte d in th e lite ra tu re [36, 38, 48], alth o u g h some co rrection is n eeded for th e presence of sm all q u a n titie s of free am ino acids d etected in soil [7]. D iffe ren t conditions of hydrolysis and an aly tic a l p ro ced u re m ay also in fluence th e results. N evertheless, th e im pression is th a t th e c o n ten t of eight am ino acids (LYS, HIS, ARG, A SP, GLU, GLY, VAL, ILEU) in th e firs t h y drolyzates of hum ic acids is com parable w ith th a t found in hydrolyzed soil. W ith resp ect to th e co n ten t of PR O and LYS soil hy d ro ly zates a re sim ilar to th e second h y dro ly zates of hum ic acids.
Obviously, conclusions of a n o v er-all n a tu re re q u ire m ore d etailed stu d y on hum ic acids deriv ed from a w id e r ran g e of soil types. O n th e basis of th e p rese n t re su lts a n assu m p tio n can be m ade th at: soil hum ic acids contain tw o m ain proteinaceo us fractio n s d ifferin g in am ino acid com position, or th e re is a d ifferen ce in th e su scep tib ility to h y d ro ly tic cleavage of p ep tid e bonds in th e am ino acid sequence of p ro tein s and peptid es w hich re su lts in tw o “fra c tio n s” havin g d iffe re n t am ino acid com position. In a n y case, am ino acid d istrib u tio n in b o th “fra c tio n s” seem s to
be re p re se n ta tiv e of proteinaceous co n stitu en ts of soil hum ic acids. T he m ore easily hy droly zed com ponents a p p e ar to be sim ilar to p la n t protein.
It is d ifficu lt to n eglect th e possibility of decyclization of N -heterocyclic non-pro teinaceou s com ponents of hum ic acids follow ed by sp littin g th em into basic and ch ain b ran ch ed am ino acids, on prolonged hydrolysis. This w ould be consistent w ith th e increase of VAL and ILEU co n ten t found in th e second h ydrolyzates. Such reactio n s w ould be expected to c o n trib u te to th e d iffe re n tia tio n of am ino acid co n tent in th e second h yd ro ly zates of hum ic acids d erived from d iffe re n t soils.
The authors are in debted to Miss Zofia Pasternak for assistance in the laboratory work.
REFERENCES
[1] A l e k s a n d r o w a L. H.: Dokł. Sow. Poczw ow iedow к VII Mieżdun. Kongr. w S. Sz. A., A. N. SSSR, I960.
[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 l a 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.
[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, W arszawa 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 S oil”, 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., N ew 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.: “Hum us”, 2nd ed., The W illiam s and W ilkins Co., Baltim o 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. CZUCHAJOW SKI, A. ERN DT
SKŁAD AMINOKWASOWY BIAŁKOWEGO KOMPONENTA KWASÓW HUMINOWYCH Z GLEBY
K atedra C hem ii O gólnej WSR w K rak ow ie
S t r e s z c z e n i e
Z gleby brunatnej w łaściw ej, w ytw orzonej z lessu (Prusy) oraz z gleby torfowej, w ytworzonej z torfu niskiego na ile m ioceńskim (Szarów), otrzymano w w yniku ekstrakcji 0,2n NaOH w tem peraturze pokojowej oraz wytrącania kwasem solnym — kwasy hum inowe I (Prusy) i II (Szarów), różniące się znacznie zawartością azotu: I — 2,77%, II — 4,00°/o. K wasy te poddano hydrolizie w warunkach beztlenowych przy udziale 6n HC1 i w otrzymanych hydrolizatach oznaczono zawartość poszczególnych am inokwasów; analizę prowadzono na autom atycznym analizatorze Beckm ann-Spinco 120B. Ponieważ hydroliza nie przeprowadziła całej ilości kw asów hum inowych do
roztworu, pozostały osad poddano powtórnej hydrolizie, po czym w e w tórnych h y drolizatach oznaczono zawartość am inokwasów. Wyniki analizy elem entarnej w y j ściowych kwasów hum inowych oraz nie zhydrolizowanych pozostałości po pierwotnej i wtórnej hydrolizie, a także ubytek zawartości azotu podano w tab. 1, zaś w yniki analizy ilościowej am inokwasów uwolnionych z części białkowej kwasów hum ino w ych w w yniku hydrolizy pierwotnej i wtórnej przedstawiono w tab. 2; przytoczone w niej wartości liczbowe podają, jaki procent azotu ogólnego, znajdującego się w e w szystkich oznaczonych am inokwasach, przypada na każdy am inokwas. W tab. 3 porównano wartości średnie poszczególnych oznaczeń z tab. 2 ze składem am inokwa- sowym białek roślinnych [46, 47] oraz zhydrolizowanych gleb [36, 38, 48].
Pierw sze hydrolizaty kwasów hum inowych obu typów — Ia i IIa — w ykazały podobny skład am inokwasowy; w w iększym stopniu różniły się jedynie zawartością ALA. Różnice składu am inokwasowego między IA i I Ia m ieściły się w granicach róż
nic znalezionych np. dla frakcji cytoplazm atycznej i chloroplastowej białka liści Phaseolus vulgaris [47]. Drugie hydrolizaty — Ib i Ив w ykazały nieco w iększe różnice składu am inokw asow ego, przekraczały one 1,5%> zawartości sumarycznego azotu am i nokw asowego w przypadku: LYS, HIS, ARG, ASP, GLU, GLY, ALA i VAL. Jednakże, co jest charakterystyczne, prawidłowość wzrostu lub spadku zawartości am inokw a sów przy przejściu od pierwotnych do wtórnych hydrolizatów okazała się dla kw a sów hum inow ych obu gleb taka sama: IIa -^ IIb , I a ^ I b . Wzrastała zawartość LYS odpowiednio o ok. 8 i 6°/o ogólnego azotu am inokwasowego, przypadającego na ten am inokwas, HIS o 7 i 3%, ARG o 9 i ll°/o, VAL o 4 i 2%> oraz ILEU o 4 i 3°/o; zm niej szyła się natom iast zawartość ASP o 4 i 7°/o i ALA o 4 i 4°/o. Charakterystyczne jest to, że skład pierwszych hydrolizatów obu kw asów hum inowych Ia i Ha znacznie bardziej przypomina skład białka roślinnego niż skład w tórnych hydrolizatów
Ib, Hb.
Można pokusić się o sugestię, że w naturalnych kwasach hum inowych istnieją dwa typy głów nych frakcji białkow ych różniących się istotnie składem am inokwa- sowym, albo też w ystępują białka, które podczas hydrolizy wykazują dość podobną od porność wiązań peptydowych, łączących określone am inokwasy, co powoduje, iż biał ka te ulegają sw ego rodzaju hydrolizie „frakcjonowanej” do dwóch frakcji, różnią cych się od siebie składem am inokwasowym.
JI. ЧУХ А Й ОВС КИ , А. ЭРНДТ АМИНОКИСЛОТНЫЙ СОСТАВ БЕЛКОВОГО КОМПОНЕНТА ГУМИНОВЫХ КИСЛОТ п о ч в ы К а ф е д р а О бщ ей Х имии, В ы сш ая С ел ь ск охозя й ств ен н ая Ш кола в К рак ов е 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. Ш а ров) были получены — в результате экстрагирования 0,2 н NaOH при комнатной температуре и осаж дения 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 содерж ание от
дельных аминокислот; анализы выполняли на автоматическом анализаторе Beckm ann-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 исходны х гуминовых кислот и негидролизуемого остатка после первого и второго гидролиза, а такж е убыль в содержании азота, поданы в таб. 1; результаты коли чественного 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 каж дую из аминокислот. В таб. 3 сравниваются средние значения отдельных определений из таб. 2 с ами нокислотным составом растительных белков (46, 47) и гидролизованных почв (36, 38, 48). Первые гидролизаты гуминовых кислот, Iа и IIа, обладали подобным амино кислотным составом; более заметно они различались единственно по содержанию ALA. Различия в аминокислотном составе м еж ду 1л и IIа находились в пределах разниц выявляемых например для цитоплазматической и хлоропластной ф рак ции белка листьев Phaseolus vulgaris [47]. Вторые гидролизаты — I/з и Ilß пока
зывали 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 повы шении 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 содерж ание 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 такж е i l e u на 4°/о и 3°/о; пони зилось ж е содерж ание 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 бли зок 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” гидро лизу этих белков до двух фракций, отличающихся друг от друга аминокилсотным составом. W płynęło do PTG w lipcu 1970 r. Dr L eszek C zu ch ajow sk i
Z akład C hem ii O gólnej U n iw e r sy te tu J a g ie llo ń sk ie g o K raków
