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Amino acid distribution in soil humic acids fractionated by tandem size exclusion chromatography polyacrylamide gel electrophoresis
EnvironmentInternational,Vol. 24, No. 5/6, pp. 573-581, 1998 Copyright©1998 ElsevierScienceLtd Printed in the USA.All fights reserved 0160-4120/98 $19.00+.00
Pergamon
PII S0160-4120(98)00036-1
AMINO ACID DISTRIBUTION IN SOIL HUMIC ACIDS FRACTIONATED BY TANDEM SIZE EXCLUSION CHROMATOGRAPHY POLYACRYLAMIDE GEL ELECTROPHORESIS O.E. Trubetskaya, O.I. Reznikova, G.V. Afanas'eva, L.F. Markova, and T.A. Muranova Branch of Shernyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 142292, Pushchino, Moscow Region, Russia
O.A. Trubetskoj Institute of Soil Science and Photosynthesis, RAS, 142292, Pushchino, Moscow Region, Russia
E1 9706-164 M (Received 27 June 1997; accepted 28 February 1998)
The objective o f this work was to study humic acids (HAs), and their fractions obtained by coupling size exclusion chromatography-polyacrylamide gel electrophoresis, from arctic soil,
podzolic soil, grey forest soil, ehemozem and red soil, in respect to their amino acids composition and content. The fractions had exactly defined molecular size (MS) and electrophoretic mobilities (EM). While HAs and fractions had remarkably similar amino acids compositions, they differed greatly in amino acids contents. The highest quantity of amino acids was found in high MS fractions (11.5-21.7%). Amino acids content essentially decreased (to 2.1-5.5%) with the increasing ofelution volumes and EM of HAs fractions, independently of HA sources. Separation of HA into less complex fractions with defined molecular and electrophoretic entities, which differ by their content of amino acids, could be of great value for studies on physical-chemical structures of HA and its genesis in soil. 01998ElsevierScienceLtd
INTRODUCTION Soil humic acids (HAs) are a diverse mixture of organic substances and consist mainly of different molecular size (MS) compounds (Schnitzer 1991). The chemical structure o f HAs is not yet completely understood, and successful fractionation into highly purified HA fractions with defined molecular entities or properties may improve knowledge o f their structural peculiarities. Taking into account that amino acids are important integral HA structural components (Stevenson 1982), energy sources for soil microorganisms, and an N source for plant growth, there have been many studies o f amino acid compositions
of HAs and humic acids fractions (HAF) obtained by different fractionation methods (Schnitzer 1985; Anderson et al. 1989). Most o f the studies have utilized size exclusion chromatography (SEC) on Sephadex as the means of MS fractionation (Swift and Posner 1972; Anderson and Hepburn 1977; Warman and Bishop 1987; Hejzlar et al. 1994). However, when using this effective technique, care must be taken in its application to HAs to ensure a genuine MS fractionation, because the results of SEC o f HAs are not always reproducible and depend on practical conditions used. Probably this is due, at least 573
574
O.E. Trubetskaya et al.
NOMENCLATURE
(Title of amino acids and their abbreviations used in the text.) Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysfeine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
Asp Thr Set Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg
partly, to the impossibility of comparing the results of HA fractionation in different conditions. Therefore, methods which can be used to compare SEC fractionation results of HAs are needed. For analysis of native biopolymers obtained by SEC, the method frequently used is polyacrylamide gel electrophoresis (PAGE) (Bath et al. 1996). Coupling SEC-PAGE should be useful for investigating the structure of HAs. However, attempts to discover the relationship between chromatographic peaks and PAGE size fractions have previously been unsuccessful (De Gonzales et al. 1981; Curvetto and Orioli 1982). A new approach has been applied to solve the problem by using PAGE in the presence of denaturing agents, as recently developed by Trubetskoj et al. (1991 ; 1992). The combination SEC-PAGE indicated a good correspondence between both fractionation systems: increasing of elution volume closely corresponds to an increase of electophoretic mobility (EM) of soil HA. Furthermore, the application of PAGE for analysis of HA material obtained by SEC allowed determination of the optimal chromatographic conditions for preparing preparative quantity highly purified HA fractions with defined molecular entities and electrophoretic properties (Yrubetskoj et al. 1997). The work reported here formed part of an extensive study on the properties of HAF obtained by tandem
SEC-PAGE. The main objectives of this portion of the study were to compare amino acid composition and content of HAs and their HAF from five soils of different origin. MATERIALS AND METHODS
The soil samples used in this study were taken from the A horizons of arctic soil (extreme north, Kolyma, Russia). podzolic soil (north of Moscow region, Russia), gray forest soil (south of Moscow region, Russia), typical chernozem (Kursk region, central part of Russia), and red soil (subtropical region, Georgia, former USSR). Some physico-chemical characteristics of the sampled soils are reported in Table 1. The HAs were obtained by extracting the soils with 0.1 M pyrophosphate and 0.1 M NaOH (pH- 13) under nitrogen gas with subsequent precipitation by HCI (pH 2.0). Compositional parameters of HAs are presented in Table 2. The fractionation of soil HA by tandem SEC-PAGE has been reported previously (Trubetskoj et al. 1997). Briefly, 10 mg of HA were dissolved by adding sufficient 0.1 M NaOH to give a solution pH approximately 7, made up to 1 mL with 7 M urea, and then dialysed against 7 M urea for 48 h and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5 x 100 cm) equilibrated with the 7 M urea. The void column volume (Vo = 47 mL) was determined using Dextran Blue 2000. Total column volume (Vt) was 160 mL. Flow rate was 20 mL/h. Column effluent was collected as 2 mL aliquots, and each third aliquot was assayed by PAGE in the presence of denaturing agents, according to Trubetskoj et al. (1991). The chromatographic aliquots, which formed an individual homogeneous electrophoretic zone in the PAGE matrix and had similar EMs, were combined into pools, dialysed against distilled water, and used for amino acids analysis. The samples (approximately 2-4 mg of HA and HAF) were hydrolysed in sealed tubes in constant boiling 5.7 N HCI for 24 h at 110°C under nitrogen. Released amino acids were injected onto a column of a cation exchange resin (automatic analyser LC 5001, Biotronic, Germany). The amino acids were eluted using a stepwise gradient of increasing pH and ionic strength. Detection was colorimetric, using post-column ninhydrin derivatisation, read at 570 nm for 16 amino acids and 440 nm for proline. Six parallel analysis of soil HA and HAF were carried out and the standard deviations calculated.
Amino acid distribution in fractionated HAs
575
Table 1. Physico-chemical properties of the soils.
Name of the soil Arctic soil Podzolic soil Gray forest soil Chernozem Red soil
Total OC g/kg
CEC meq/100 g
Exchangeable basis meq/100 g
pH
n.d.* 10.0 19.2 49.0 17.1
n.d. 8.0 18.5 45.2 0.3
4.4 5.8 6.9 7.5 4.5
267 13 11 48 26
* n.d. - Not determined.
Table 2. Compositional parameters of soil HAs.
Name of soil HA Arctic soil HA Podzolic soil HA Gray forest soil HA Chemozem HA Red soil HA
C g/kg HA
H g/kg HA
N g/kg HA
n.d. 576 612 626 598
n.d. 52 36 28 42
n.d. 48 39 32 45
RESULTS AND DISCUSSION
In this study, five HAs, originated from different soils, were carefully fractionated by SEC in 7 M urea as eluent (Fig. 1). The shapes of the different curves had some differences, but all HA material, applied on the column, eluted within the total column volume without any adsorption on the Sephadex gel matrix. Column effluent was collected as chromatographic aliquots, and each third aliquot was assayed by PAGE in the presence of denaturing agents. Those aliquots which formed an individual homogeneous electrophoretic zone in the PAGE matrix combined into HAF "A", "B", and "C+D". For all HA investigated, fractions "A" corresponded to the excluded peaks (elution volume (Ve) was 47-55 mL) formed on the electropherograms start zone that did not move into the 10% PAGE. Fractions "B" (Ve=58-80 mL) and "C+D" (Ve=110-150 mL) were collected in the fractionation range of the column. Fraction "B" formed one intensely colored zone in the mid part of the 10% PAGE slab. The combined fraction "C+D" formed several intensely colored bands with relatively close EM in the bottom of the PAGE slab. Identically marked fractions had similar EM independently of HA sources (Figs. 2 and 3). As elution volumes were similar for chromatographic pools, which formed bands with identical EM, it can be sug-
Ash g/kg HA 13 31 17 22 95
gested that they have similar MS, and MS of fraction "A" > MS of fraction "B" > MS of fraction "C+D". Amino acids compositions of HA and HAF from soils investigated are shown in Tables 3-7. All samples contained 23-32% of acidic (Glu+Asp), 11-17% of basic (Lys+Arg+His), 20-29% of neutral hydrophobic (Pro+Val+Ile+Leu+Phe), and 33-40% of neutral hydrophilic (Thr+Ser+Gly+Ala) amino acids. The most abundant amino acids were Asp, Glu, Gly, and Ala. These observations could indicate that amino acids and peptide residues in all of the fractions had similar origins. On the other hand, all HA samples and HAF differed greatly in total amino acids content (Tables 3-8). The smallest content of amino acids was found in a gray forest soil and chemozem HA (5.9% and 6.1%, respectively), derived from soils with similar genesis (Kononova 1966). Red soil HA, derived from subtropical soil, contained 8.0% of amino acids, while in podzolic soil and arctic soil, HA amino acids contents were 11.3% and 16.3%, respectively. The highest amino acids content was found in high MS fractions "A" (excluded peaks), which changed from 11.5 to 13.9% in gray forest soil, red soil, chernozem, and podzolic soil, respectively, and increased to 21.7% in arctic soil. Amino acids content essentially decreased with increasing elution volumes and EMs of HAF investigated. Fractions "B" from all
576
O.E. Trubetskaya et al.
A254
0.5-
Arctic soil I-LA
X i
t
I
i
I
I
Podzolic soil I-L~
(1)
|
(2)
0.5-
i
I
i
i
!
G r a y forest soil HA
I
(3)
0.5-
I
I
I
J
i
Chernozem tL~
I
(4)
0.5-
i
I
!
I
I
Red soil HA
i
(5)
0.5-
A
0
I',
Vo 60
C+D
B I
120
[ I
Vt
!
180 mL
Fig. 1. SEC of 10 mg HA from arctic soil (1), podzolic soil (2), gray forest soil (3), chemozem (4), and red soil (5) on Sephadex G-75 column (100xl.5 cm) using 7 M urea as eluting system. Black boxes on the X-axis show the combined fractions "A", "B", and "C+D". obtained on the basis of electrophoretic analysis.
Amino acid distribution in fractionated HAs
577
1
2
3
4
5
A
B
C+D
Fig. 2. Electrophoresis of 0.25 mg HA from arctic soil (1), podzolic soil (2), gray forest soil (3), chemozem (4), and red soil (5) in 10% polyacrylamide gel in the presence of denaturing agents.
1
2
3
1
2
3
1
2
3
A
B
C+D
Fig. 3. Electrophoresis of 0.1 mg combined fractions "A", "B", and "C+D" from gray forest soil (1), chemozem (2), and red soil (3) obtained aider SEC. Po,dzolic soil and arctic soil HA fractions show the same patterns on the eleetropherograms.
578
O.E. Trubetskaya et al.
Table 3. Amino acids released after hot acid hydrolysis of arctic soil HA and fractions"A","B", and"C+D", obtained by tandem SEC-PAGE HA
A
1'
2**
3***
r3
1
2
3
C+D
I
2
3
I
2
Asp
170.2±6.4
13.2
22.7
200.4±24.1
12.2
26.7
86.0±2.0
13.7
I 1.4
28.4±4.0
12.6
3.8
Thr
99.6±0.9
7.7
11.9
I09.5±13.1
6.7
13.0
44.3±1.2
7.0
5.3
15.8±25
7.0
19
Ser
74.0±1.7
5.7
7.6
81.4±10.0
5.0
8.4
41.8±1.3
6.6
4.3
18.4±3.4
8.1
I9
Glu
130.4~2.5
10.1
19.2
214.0±25.3
13.0
31.5
63.8±2.3
10.1
9.4
23.2±3.4
10.3
34
Pro"
82.2:kl .7
6.4
9.5
92.9±8.9
5.7
10.7
41.5±1.2
6.6
4.8
I 1.6±0.9
5. l
I
Gly
164.3±6.6
12.7
12.3
143.4±18.1
8.7
10.8
89.9±2.4
14.3
6.7
35.3±5.6
15.6
2.6
Ala
131.1±1.8
10.2
11.7
180.5±23.0
11.0
16.1
63.1~:1.9
10.0
10.2
Cys
4.4±0.9
0.5
35.1±7.0
4.3
4.1±0.4
Val
95.9±0.8
7,4
11.2
127.5±16.3
14.9
43.0±1.4
Met
8.7±1.0
7.8
6.8
5.6
23~1±3.5
0.5
traces
5.0
14.4±2.3
2, t trace~
6.4
17
1.3
15.4±2.1
2.3
4.8±0.4
0.7
traces
Ile
60.3±2.6
4.7
7.9
58.7±6.2
3.6
7.7
27.1±1.0
4.3
3.6
9.6±1.2
4.2
traces
Leu
80.9±3.1
6.3
10.6
126.6±18.4
7.7
16.6
39.4±1.2
6.3
5.2
13.7±2.1
6.1
Tyr
24.9±0.3
4.5
31.2+4.2
5.7
11.3±0.2
2.0
4.4±0.6
Phe
49.6±1.4
3.8
8.2
75.5+10.3
4.6
12.5
22.1±0.3
3.5
3.7
8.6±1.5
3,8
1.4
His
51,4±1.9
4.0
8.0
61.6±9.2
3.8
9.6
23.1±1.1
3.7
3.6
I I.lxl.3
4,9
17
Lys
54,0±1.1
4.2
7.9
105.84-12.9
6.5
15.5
23.4±2.8
3.7
3.4
7.7±1.3
3.4
il
Arg
43,0±2.6
3.3
7.5
60.5±6.8
3.7
10.5
17.0±1.1
2.7
3.0
5.2±0.6
2.3
0.9
13 1.8 08
* nmol/mg - nmol of amino acid per nag of dr 5, HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~2nmol/mg of all amino acids. C~,s, Met, and I y r do not take into account: *** mg/g - mg of amino acid per gram of dry HA sample.
Table 4. Amino acids released after hot acid hydrolysis of podzolic soil HA and fractions "A", "B", and " C + D ' , obtained by tandem SEC-PAGE. HA
Asp
A
B
C+D
l*
2**
3***
1
2
3
1
2
3
1
2
3
142.24-23.3
15.6
18.9
168.8+9.9
14.6
22.5
142.3±40.7
15.6
18.9
48.9+5.0
13.5
6.5
Thr
55.84-10.1
6.1
6.6
73.7+0.6
6.4
8.8
54.8±16.4
6.0
6.5
19.9+1.6
5.5
2,4
Ser
42.9a:7.7
4.7
4.4
59.2+1.7
5.1
6.1
44.7±10.3
4.9
4.6
20.4±2.3
5.7
21
Glu
103.7±19.5
11.4
15.3
135.3:k5.0
11.7
19.9
113.4+30.9
12.4
16.7
49.0+4.2
13.6
7.2
Pro
54.84-9.7
6.0
6.3
66.6+1.6
5.8
7.7
55.8±I 1.5
6. I
6.4
17.3±2.9
4.8
2.(I 4.2
Gly
127.54-21.7
14.0
9.6
161.9±6.5
14.0
12.2
128.8±36.0
14.1
9.7
56.4+6.1
15.7
Ala
87.84-15.8
9.7
7.8
116.84-0.3
10.1
10.4
84.5±25.9
9.2
7,5
31.6±4.2
8.8
Cys
2.94-0.6
0.4
6.0±2.3
0.7
4.7±0.4
0.6
traces
Val
62.74-11.2
9.9
60.9±19. l
7.1
20.6±2.3
Met
5.1±0.7
1.7
6.5±2.5
1.0
2.6±0.9
Ile
37.74-6.7
Leu
50.64-9.0
6.9
7.3
6.7
2,8 traces
7.3
84.64-1.3
0.8
11.6±4.0
5.7
2.4
4.1
4.9
51.1+0.7
4.4
6.7
36.9+10.9
4.0
4.8
13.9±2.0
3.9
1.8
5.6
6.6
72.44-1.2
6.3
9.5
50.0+13.1
5.5
6.6
19.9±1.9
5.5
2.6
04
Tyr
8.1±1.4
1.5
9.5±0.8
1.7
8.6±1.9
1.6
4.6±1.5
Phe
28.14-5.2
3.1
4.6
37.9+1.8
3.3
6.3
28.0±7.4
3.t
4.6
11.1±1.3
3.1
0.8 1.8
His
34.2±6.3
3.8
5.3
45.8±6.0
4.0
7.1
32.8±8.5
3.6
5.1
15.9±1.7
4.4
2.5
Lys
55.5±10.1
6.1
8.1
51.0±3.0
4.4
7.5
53.7±13.0
5.9
7.9
19.84-2.1
5.5
2.9
Arg
25.8±4.7
2.8
4.5
29.6±2.5
2.6
5.2
27.6±6.1
3.0
4.8
10.6±1.7
2.9
1.8
* nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg o f each amino acid)/~nmol/mg of all amino acids, Cys, Met, and I y r do not take into account; *** mg/g - mg of amino acid per gram of dry HA sample.
Amino acid distribution in fractionated HAs
579
Table 5. Amino acids released after hot acid hydrolysis of gray forest soil HA and fractions "A", "B", and "C+D", obtained by tandem SEC-PAGE. HA 1"
2**
A 3***
1
B 2
3
1
C+D 2
3
1
2
3
Asp 64.0-J:17.8 13.9 8.5 111.2:t:61.6 12.4 14.8 93.2-4-28.0 14.6 12.4 29.9-±2.4 18.5 4.0 Thr 23.7±7.5 5.2 2.8 57.14-32.4 6.4 6.8 35.44-10.5 5.6 4.2 7.2±0.6 4.5 0.9 Ser 22.14-7.6 4.8 2.3 51.44-26.8 5.7 5.3 36.34-9.1 5.7 3.7 9.14-1.4 5.6 0.9 Glu 50.84-13.4 11.0 7.5 94.74-49.2 10.6 13.9 78.4±22.0 12.3 11.5 16.9-±1.6 10.5 2.5 Pro 33.84-7.8 7.5 3.9 45.4±22.5 5.1 5.2 34.6±12.3 5.4 4.0 7.0-±0.5 4.3 0.8 Gly 59.94-17.1 13.0 4.5 120.7±66.5 13.5 9.1 92.9-±25.1 14.6 7.0 26.1±2.9 16.2 2.0 Ala 38.64-11.0 8.4 3.8 91.7±50.2 10.2 9.1 59.8±16.5 9.4 5.9 12.2±1.8 7.5 1.2 Cys 2.34-0.6 0.3 6.3±3.2 0.8 4.6±1.0 0.6 traces traces Val 25.9-a:15.3 5.6 3.0 61.5±33.5 6.9 7.2 35.54-10.3 5.6 4.2 7.4±0.8 4.6 0.9 Met 2.44-0.6 0.4 9.7±7.6 1.4 8.7±3.5 1.3 1.94-0.2 0,3 Ile 19.04-2.1 4.1 2.5 34.94-25.5 3.9 4.6 18.3±7.6 2.9 2.4 4.8±0.2 3.0 0.6 Leu 29.84-3.6 6.5 3.9 58.8±42.4 6.6 7.7 27.8±11.6 4.4 3.6 7.94-0.1 4.9 1.0 Tyr 5.04-2.0 0.9 9.64-5.6 1.7 4.4±0.5 0.8 1.8±0.1 0.3 Phe 15.54-4.6 3.4 2.6 30.6±14.9 3.4 5.1 17.24-5.2 2.7 2.8 4.5±0.2 2.8 0.7 His 27.74-10.3 6.0 4.3 49.54-26.8 5.5 7.7 37.54-9.9 5.9 5.8 8.0±1.2 5.0 1.2 Lys 31.04-7.9 6.7 4.5 43.3±1.4 4.8 6.3 42.24-13.2 6.6 6.2 13.8±0.8 8.5 2.0 Arg 18.1±4.8 3.9 3.2 44.6±5.7 5.0 7.8 28.7±7.1 4.5 5.0 6.8±2.0 4.2 1.2 * nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~nmoi/mg of all amino acids, Cys, Met, and Tyr do not take into account; *** mg/g - mg of amino acid per gram of dry HA sample.
Table 6. Amino acids released after hot acid hydrolysis of chemozem HA and fractions "A", "B", and "C+D", obtained by tandem SECPAGE. HA 1'
2**
A 3***
1
B 2
3
1
Asp 81.7+11.3 17.0 10.9 187.94-49.9 18.1 25.0 118.6+8.0 Thr 22.7±3.3 4.7 2.7 52.8±8.5 5.1 6.3 29.9±0.3 Ser 20.9±3.1 4.4 2.2 47.8±4.2 4.6 4.9 29.6±4.2 Glu 55.5+7.2 11.6 8.2 121.2±16.1 11.6 17.8 81.1±16.0 Pro 33.4+3.5 7.0 3.8 47.5±2.0 4.6 5.5 35.0±6.2 Gly 65.4+8.8 13.6 4.9 145.8±33.1 14.0 10.9 90.4±5.7 Ala 39.4±6.3 8.2 3.9 102.24-26.3 9.8 10.1 54.4±1.3 Cys 2.2±0.6 0.3 10.74-2.2 1.3 5.54-0.5 Val 27.6±3.9 5.8 3.2 69.4+15.6 6.7 8.1 36.0~1.2 Met 2.14-0.2 0.3 7.94-5.5 1.2 5.24-2.9 lie 19.14-1.9 4.0 2.5 40.9±6.6 3.9 5.4 18.54-1.8 Leu 28.2±2.9 5.9 3.7 66.04-13.1 6.3 8.7 27.5±2.5 Tyr 4.8±0.5 0.9 7.1±2.1 1.3 3.94-1.7 Phe 14.6a:2.0 3.0 2.4 30.54-3.4 2.9 5.0 16.04-2.8 His 26.0-±5.1 5.4 4.0 44.1±8.1 4.2 6.8 28.1±0.3 Lys 28.1±3.5 5.9 4.1 47.3±1.6 4.5 6.9 35.8±5.4 Arg 16.84-2.0 3.5 2.9 37.34-9.5 3.6 6.5 22.0-±8.7 * nmol/mg - nmol of amino acid per mg of dry HA sample; ** n m o l % - 100(nmol/mg of each amino acid)/~nmol/mg of all amino acids, Cys, *** mg/g - m g of amino acid per gram of dry HA sample.
C+D 2
3
1
2
3
19.0 4.8 4.8 13.0 5.6 14.5 8.7
15.8 3.6 3.1 11.9 4.0 6.8 5.4 0.7 4.2 0.8 2.4 3.6 0.7 2,6 4.4 5.2 3.8
31.4±3.8 8.1±0.3 9.4±2.1 21.1+3.3 10.5±1.8 30.7±3.2 14.74-0.1 traces 9.34-0.1 1.7±0.3 5.0-±0.3 7.94-1.2 1.5±0.7 5.6±0.4 9.5±0.8 11.4±2.5 9.4±3.9
17.1 4.4 5.1 11.5 5.7 16.7 8.0
4.2 1.0 1.0 3.1 1.2 2.3 1.5 traces 1.1 0.3 0.7 1.0 0.3 0.9 1.5 1.7 1.6
5.8 3.0 4.4 2.6 4.5 5.7 3.5
5.1 2.7 4.3 3.0 5.2 6.2 5.1
Met, and Tyr do not take into account;
580
O.E. Trubetskaya et al.
Table 7. Amino acids released after hot acid hydrolysis of red soil HA and fractions " A ' , "B", and "C+D", obtained by tandem SEC-PAGE. HA 1'
A
B
C+D
2**
3***
1
2
3
I
2
3
I
2
Asp 82.2+4.0 Thr 39.6+1.3 Ser 35.8+3.3
13.0
10.9
140.0+15.5
13.8
18.6
126.9+20.9
14.4
16.9
68.8+26.6
15.4
9.2
6.2
4.7
68.6+6.3
6.8
8.2
55.1±8.9
6.2
6.6
22.8+8.1
5.1
2.7
5.6
3.7
66.8+6.6
6.6
6.9
52.0+7.3
5.9
5.4
25.3±6.8
5.6
2.~
Glu
66.5+3.6
10.5
9.8
104.2+12.4
10.3
15.3
106.6±16.8
12.1
15.7
48.6+12.6
10.8
7.2
Pro
53.7+5.9
8.5
6.2
62.1+10.5
6.1
7.1
60.8+6 8
6.9
7,0
5.9
3.(i
Gly
79.5+6.3
12.5
6.0
141.8+14.2
14.0
10.6
127.9+20.2
14.5
9.6
78.8+23.8
17.6
5.9
Ala
53.9+3.0
8.5
5.3
102.4+8.8
10.1
10.1
82.9+14.6
9.4
8.2
38.7+14.2
8.6
3.8
Cys 2.1±0.1 Val 40.8±0.4 Met 1.9+0.9
0.3
6.9+t:3.7
0.8
6.3±1.6
6.4
4.8
70.2+7.4
6.9
8.2
54.2±6.8
0.3
7.0~1.6
-
1.0
5.9±22
Ile
26.1+2.2
4.1
3.4
35.7+4.7
3.5
4.7
30.0±3.4
3.4
3.9
15.8+3.5
3.5
Leu 40.7±4.5
6.4
5.3
56.0+8,6
5.5
7.3
45.8+4.0
5.2
6.0
24.5+4.7
5.5
1.4
11.7:~2.0
2.1
8.3±2.1
1,5
5.1±1.6
Tyr
8.0+1.1
Phe 26.2±1.3 His 39.1+5.0
6.1
26.3t7.9
0.8
traces
6.3
23.9+7.7
0.9
1.9+0.7
traces 5.3
2.8 0.3 2.1 3.2 0.9
4.1
4.3
40.6:L9.0
4.0
6.7
28.4±3.8
3.2
4.7
14.8+3.8
3.3
2.4
6.2
6.1
61.7+10.4
6.1
9.6
46.6±6.0
5.3
7.2
23.5±5.9
5.2
3.6
30.4+1.4
4.8
4.4
38.4+4.2
3.8
5.6
41.4+5.6
4.7
6.1
20.5±3.8
4.6
3.{)
Arg 20.2+1.9
3.2
3.5
26.3±2.8
2.6
4.6
25.4±8.5
2.9
4.4
15.7+2.7
3.5
2.7
Lys
* nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~nmol/mg of all amino acids, Cys, Met, and Tyr do not take into account: *** mg/g - m g of amino acid per gram of dry HA sample.
Table 8. Amino acids weight content (%) in five soil HAs of different origin and fractions "'A", "B", and "C+D", obtained by SEC-PAGE.
Name of the soil Arctic soil Podzolic soil Gray forest soil Chernozem Red soil
HA
A
B
C+D
16.3
21.7
7.8
2.8
11.3
13.9
I 1.5
4.4
5.9
11,5
8. I
2.1
6.1
13,2
7.9
2.3
8.0
12,7
11.1
5.5
soils investigated contained from 7.5 to 11.5% of amino acids. The smallest amino acids content, from 2.1 to 5.5%, was found in low MS fractions "C+D". The larger content of amino acids from high MS fractions may be due to the hydrolysis of peptide of relatively greater chain length.
CONCLUSION
The five HAs from soils of different genesis and climatic conditions were fractionated by coupling SECPAGE. Preparative quantities of three highly purified
HA fractions ("A", "B", and "C+D") with defined molecular and electrophoretic entities from each soil HA sample were obtained. Amino acids contents of each HA and its fractions were studied. The highest amino acids content (11.5-21.7%) was found in high MS fractions "A" and the smallest amino acids content (2.1-5.5%) was found in low MS fractions "C+D". If the structural complexity of humic macromolecules is taken into account, a separation of HA into less complex fractions with defined molecular and electrophoretic entitles, which differ by their content of amino acids, could be of great value for studies of physicalchemical structures of HA and its genesis in soil.
Amino acid distribution in fractionated HAS
REFERENCES Anderson, H.A.; Hepburn, A. Fractionation of humic acid by gel permeation chromatography. 3. Soil Sci. 28: 634-644; 1977. Anderson, H.A.; Bick, W.; Hepburn, A.; Stewart, M. Nitrogen in humic substances. In: Hayes, M.H.B.; MacCarthy, P.; Malcolm, R.L.; Swift R.S., eds. Humic substances II. In search of structure. Chichester: John Wiley and Sons; 1989: 223-253. Bath, H.G.; Boyes, B.E.; Jackson, C. Size exclusion chromatography. Anal. Chem. 68: 445R-466R, 1996. Curvetto, N.R.; Orioli, G.A. Electrophoretic subfractionation of low and high molecular weight humic acid fractions. Plant Soil 66: 205-215; 1982. De Gonzales, N.M.; Castagnola, M.; Rossetti, D. Humic acid characterization of Colombian soil by disc electrophoresis and infrared spectroscopy following gel filtration. J. Chromatogr. 209: 421-431; 1981. Hejzlar, J.; Szpakowska, B.; Wershaw, R.L. Comparison of humic substances isolated from peatbog water by sorption on DEAEcellulose and Amberlite XAD-2. Water Res. 28: 1969-1970; 1994. Kononova, M.M. Soil organic matter. Elmsford: Pergamon Press; 1966. Schnitzer, M. Nature of nitrogen in humic substances. In: Hayes, M.H.B.; Aiken, G.R.; M&night, P.M.; Wershaw, R.L.;
581
Mac&thy, P., eds. Humic substances in soil, sediment and water. Chichester: John Wiley; 1985: 303-325. Schnitzer, M. Soil organic matter - The next 75 years. Soil Sci. 151: 41-58; 1991. Stevenson, F.G. Humus chemistry: Genesis, composition, reactions. New York, NY: John Wiley & Sons; 1982. Swift, R.S.; Posner, A.M. Nitrogen, phosphorus and sulphur contents of humic acids fractionated with respect to molecular weight. J. Soil Sci. 23: 50-57; 1972. Trubetskoj, O.A.; Kudryavceva, L.Y.; Shirshova, L.T. Characterization of soil humic matter by polyacrylamide gel electrophoresis in the presence of denaturating agents. Soil Biol. Biochem. 23: 1179-1181; 1991. Trubetskoj, O.A.;Trubetskaya, O.E.; Khonutova, T.E. Isolation, purification, and some physico-chemical properties of soil humic substances fractions obtained by polyacrylamide gel electrophoresis. Soil Biol. Biochem. 24: 893-896; 1992. Trubetskoj, O.A.; Trubetskaya, O.E.; Afanas’eva, G.V.; Reznikova, 0.1.; Saiz-Jimenez, C. Polyacrylamide gel electrophoresis of soil humic acid fractionated by size-exclusion chromatography and ultrafiltration. J. Chromatogr. A 767: 285-292; 1997. Warman, P.R.; Bishop, C. Amino-N compounds found in soil organic matter hydrolysates of loamy sand using an immobilized protease reactor column. Biol. Fertility Soils 5: 219224; 1987.
Pergamon
PII S0160-4120(98)00036-1
AMINO ACID DISTRIBUTION IN SOIL HUMIC ACIDS FRACTIONATED BY TANDEM SIZE EXCLUSION CHROMATOGRAPHY POLYACRYLAMIDE GEL ELECTROPHORESIS O.E. Trubetskaya, O.I. Reznikova, G.V. Afanas'eva, L.F. Markova, and T.A. Muranova Branch of Shernyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, 142292, Pushchino, Moscow Region, Russia
O.A. Trubetskoj Institute of Soil Science and Photosynthesis, RAS, 142292, Pushchino, Moscow Region, Russia
E1 9706-164 M (Received 27 June 1997; accepted 28 February 1998)
The objective o f this work was to study humic acids (HAs), and their fractions obtained by coupling size exclusion chromatography-polyacrylamide gel electrophoresis, from arctic soil,
podzolic soil, grey forest soil, ehemozem and red soil, in respect to their amino acids composition and content. The fractions had exactly defined molecular size (MS) and electrophoretic mobilities (EM). While HAs and fractions had remarkably similar amino acids compositions, they differed greatly in amino acids contents. The highest quantity of amino acids was found in high MS fractions (11.5-21.7%). Amino acids content essentially decreased (to 2.1-5.5%) with the increasing ofelution volumes and EM of HAs fractions, independently of HA sources. Separation of HA into less complex fractions with defined molecular and electrophoretic entities, which differ by their content of amino acids, could be of great value for studies on physical-chemical structures of HA and its genesis in soil. 01998ElsevierScienceLtd
INTRODUCTION Soil humic acids (HAs) are a diverse mixture of organic substances and consist mainly of different molecular size (MS) compounds (Schnitzer 1991). The chemical structure o f HAs is not yet completely understood, and successful fractionation into highly purified HA fractions with defined molecular entities or properties may improve knowledge o f their structural peculiarities. Taking into account that amino acids are important integral HA structural components (Stevenson 1982), energy sources for soil microorganisms, and an N source for plant growth, there have been many studies o f amino acid compositions
of HAs and humic acids fractions (HAF) obtained by different fractionation methods (Schnitzer 1985; Anderson et al. 1989). Most o f the studies have utilized size exclusion chromatography (SEC) on Sephadex as the means of MS fractionation (Swift and Posner 1972; Anderson and Hepburn 1977; Warman and Bishop 1987; Hejzlar et al. 1994). However, when using this effective technique, care must be taken in its application to HAs to ensure a genuine MS fractionation, because the results of SEC o f HAs are not always reproducible and depend on practical conditions used. Probably this is due, at least 573
574
O.E. Trubetskaya et al.
NOMENCLATURE
(Title of amino acids and their abbreviations used in the text.) Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysfeine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine
Asp Thr Set Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg
partly, to the impossibility of comparing the results of HA fractionation in different conditions. Therefore, methods which can be used to compare SEC fractionation results of HAs are needed. For analysis of native biopolymers obtained by SEC, the method frequently used is polyacrylamide gel electrophoresis (PAGE) (Bath et al. 1996). Coupling SEC-PAGE should be useful for investigating the structure of HAs. However, attempts to discover the relationship between chromatographic peaks and PAGE size fractions have previously been unsuccessful (De Gonzales et al. 1981; Curvetto and Orioli 1982). A new approach has been applied to solve the problem by using PAGE in the presence of denaturing agents, as recently developed by Trubetskoj et al. (1991 ; 1992). The combination SEC-PAGE indicated a good correspondence between both fractionation systems: increasing of elution volume closely corresponds to an increase of electophoretic mobility (EM) of soil HA. Furthermore, the application of PAGE for analysis of HA material obtained by SEC allowed determination of the optimal chromatographic conditions for preparing preparative quantity highly purified HA fractions with defined molecular entities and electrophoretic properties (Yrubetskoj et al. 1997). The work reported here formed part of an extensive study on the properties of HAF obtained by tandem
SEC-PAGE. The main objectives of this portion of the study were to compare amino acid composition and content of HAs and their HAF from five soils of different origin. MATERIALS AND METHODS
The soil samples used in this study were taken from the A horizons of arctic soil (extreme north, Kolyma, Russia). podzolic soil (north of Moscow region, Russia), gray forest soil (south of Moscow region, Russia), typical chernozem (Kursk region, central part of Russia), and red soil (subtropical region, Georgia, former USSR). Some physico-chemical characteristics of the sampled soils are reported in Table 1. The HAs were obtained by extracting the soils with 0.1 M pyrophosphate and 0.1 M NaOH (pH- 13) under nitrogen gas with subsequent precipitation by HCI (pH 2.0). Compositional parameters of HAs are presented in Table 2. The fractionation of soil HA by tandem SEC-PAGE has been reported previously (Trubetskoj et al. 1997). Briefly, 10 mg of HA were dissolved by adding sufficient 0.1 M NaOH to give a solution pH approximately 7, made up to 1 mL with 7 M urea, and then dialysed against 7 M urea for 48 h and loaded onto a Sephadex G-75 (Pharmacia, Sweden) column (1.5 x 100 cm) equilibrated with the 7 M urea. The void column volume (Vo = 47 mL) was determined using Dextran Blue 2000. Total column volume (Vt) was 160 mL. Flow rate was 20 mL/h. Column effluent was collected as 2 mL aliquots, and each third aliquot was assayed by PAGE in the presence of denaturing agents, according to Trubetskoj et al. (1991). The chromatographic aliquots, which formed an individual homogeneous electrophoretic zone in the PAGE matrix and had similar EMs, were combined into pools, dialysed against distilled water, and used for amino acids analysis. The samples (approximately 2-4 mg of HA and HAF) were hydrolysed in sealed tubes in constant boiling 5.7 N HCI for 24 h at 110°C under nitrogen. Released amino acids were injected onto a column of a cation exchange resin (automatic analyser LC 5001, Biotronic, Germany). The amino acids were eluted using a stepwise gradient of increasing pH and ionic strength. Detection was colorimetric, using post-column ninhydrin derivatisation, read at 570 nm for 16 amino acids and 440 nm for proline. Six parallel analysis of soil HA and HAF were carried out and the standard deviations calculated.
Amino acid distribution in fractionated HAs
575
Table 1. Physico-chemical properties of the soils.
Name of the soil Arctic soil Podzolic soil Gray forest soil Chernozem Red soil
Total OC g/kg
CEC meq/100 g
Exchangeable basis meq/100 g
pH
n.d.* 10.0 19.2 49.0 17.1
n.d. 8.0 18.5 45.2 0.3
4.4 5.8 6.9 7.5 4.5
267 13 11 48 26
* n.d. - Not determined.
Table 2. Compositional parameters of soil HAs.
Name of soil HA Arctic soil HA Podzolic soil HA Gray forest soil HA Chemozem HA Red soil HA
C g/kg HA
H g/kg HA
N g/kg HA
n.d. 576 612 626 598
n.d. 52 36 28 42
n.d. 48 39 32 45
RESULTS AND DISCUSSION
In this study, five HAs, originated from different soils, were carefully fractionated by SEC in 7 M urea as eluent (Fig. 1). The shapes of the different curves had some differences, but all HA material, applied on the column, eluted within the total column volume without any adsorption on the Sephadex gel matrix. Column effluent was collected as chromatographic aliquots, and each third aliquot was assayed by PAGE in the presence of denaturing agents. Those aliquots which formed an individual homogeneous electrophoretic zone in the PAGE matrix combined into HAF "A", "B", and "C+D". For all HA investigated, fractions "A" corresponded to the excluded peaks (elution volume (Ve) was 47-55 mL) formed on the electropherograms start zone that did not move into the 10% PAGE. Fractions "B" (Ve=58-80 mL) and "C+D" (Ve=110-150 mL) were collected in the fractionation range of the column. Fraction "B" formed one intensely colored zone in the mid part of the 10% PAGE slab. The combined fraction "C+D" formed several intensely colored bands with relatively close EM in the bottom of the PAGE slab. Identically marked fractions had similar EM independently of HA sources (Figs. 2 and 3). As elution volumes were similar for chromatographic pools, which formed bands with identical EM, it can be sug-
Ash g/kg HA 13 31 17 22 95
gested that they have similar MS, and MS of fraction "A" > MS of fraction "B" > MS of fraction "C+D". Amino acids compositions of HA and HAF from soils investigated are shown in Tables 3-7. All samples contained 23-32% of acidic (Glu+Asp), 11-17% of basic (Lys+Arg+His), 20-29% of neutral hydrophobic (Pro+Val+Ile+Leu+Phe), and 33-40% of neutral hydrophilic (Thr+Ser+Gly+Ala) amino acids. The most abundant amino acids were Asp, Glu, Gly, and Ala. These observations could indicate that amino acids and peptide residues in all of the fractions had similar origins. On the other hand, all HA samples and HAF differed greatly in total amino acids content (Tables 3-8). The smallest content of amino acids was found in a gray forest soil and chemozem HA (5.9% and 6.1%, respectively), derived from soils with similar genesis (Kononova 1966). Red soil HA, derived from subtropical soil, contained 8.0% of amino acids, while in podzolic soil and arctic soil, HA amino acids contents were 11.3% and 16.3%, respectively. The highest amino acids content was found in high MS fractions "A" (excluded peaks), which changed from 11.5 to 13.9% in gray forest soil, red soil, chernozem, and podzolic soil, respectively, and increased to 21.7% in arctic soil. Amino acids content essentially decreased with increasing elution volumes and EMs of HAF investigated. Fractions "B" from all
576
O.E. Trubetskaya et al.
A254
0.5-
Arctic soil I-LA
X i
t
I
i
I
I
Podzolic soil I-L~
(1)
|
(2)
0.5-
i
I
i
i
!
G r a y forest soil HA
I
(3)
0.5-
I
I
I
J
i
Chernozem tL~
I
(4)
0.5-
i
I
!
I
I
Red soil HA
i
(5)
0.5-
A
0
I',
Vo 60
C+D
B I
120
[ I
Vt
!
180 mL
Fig. 1. SEC of 10 mg HA from arctic soil (1), podzolic soil (2), gray forest soil (3), chemozem (4), and red soil (5) on Sephadex G-75 column (100xl.5 cm) using 7 M urea as eluting system. Black boxes on the X-axis show the combined fractions "A", "B", and "C+D". obtained on the basis of electrophoretic analysis.
Amino acid distribution in fractionated HAs
577
1
2
3
4
5
A
B
C+D
Fig. 2. Electrophoresis of 0.25 mg HA from arctic soil (1), podzolic soil (2), gray forest soil (3), chemozem (4), and red soil (5) in 10% polyacrylamide gel in the presence of denaturing agents.
1
2
3
1
2
3
1
2
3
A
B
C+D
Fig. 3. Electrophoresis of 0.1 mg combined fractions "A", "B", and "C+D" from gray forest soil (1), chemozem (2), and red soil (3) obtained aider SEC. Po,dzolic soil and arctic soil HA fractions show the same patterns on the eleetropherograms.
578
O.E. Trubetskaya et al.
Table 3. Amino acids released after hot acid hydrolysis of arctic soil HA and fractions"A","B", and"C+D", obtained by tandem SEC-PAGE HA
A
1'
2**
3***
r3
1
2
3
C+D
I
2
3
I
2
Asp
170.2±6.4
13.2
22.7
200.4±24.1
12.2
26.7
86.0±2.0
13.7
I 1.4
28.4±4.0
12.6
3.8
Thr
99.6±0.9
7.7
11.9
I09.5±13.1
6.7
13.0
44.3±1.2
7.0
5.3
15.8±25
7.0
19
Ser
74.0±1.7
5.7
7.6
81.4±10.0
5.0
8.4
41.8±1.3
6.6
4.3
18.4±3.4
8.1
I9
Glu
130.4~2.5
10.1
19.2
214.0±25.3
13.0
31.5
63.8±2.3
10.1
9.4
23.2±3.4
10.3
34
Pro"
82.2:kl .7
6.4
9.5
92.9±8.9
5.7
10.7
41.5±1.2
6.6
4.8
I 1.6±0.9
5. l
I
Gly
164.3±6.6
12.7
12.3
143.4±18.1
8.7
10.8
89.9±2.4
14.3
6.7
35.3±5.6
15.6
2.6
Ala
131.1±1.8
10.2
11.7
180.5±23.0
11.0
16.1
63.1~:1.9
10.0
10.2
Cys
4.4±0.9
0.5
35.1±7.0
4.3
4.1±0.4
Val
95.9±0.8
7,4
11.2
127.5±16.3
14.9
43.0±1.4
Met
8.7±1.0
7.8
6.8
5.6
23~1±3.5
0.5
traces
5.0
14.4±2.3
2, t trace~
6.4
17
1.3
15.4±2.1
2.3
4.8±0.4
0.7
traces
Ile
60.3±2.6
4.7
7.9
58.7±6.2
3.6
7.7
27.1±1.0
4.3
3.6
9.6±1.2
4.2
traces
Leu
80.9±3.1
6.3
10.6
126.6±18.4
7.7
16.6
39.4±1.2
6.3
5.2
13.7±2.1
6.1
Tyr
24.9±0.3
4.5
31.2+4.2
5.7
11.3±0.2
2.0
4.4±0.6
Phe
49.6±1.4
3.8
8.2
75.5+10.3
4.6
12.5
22.1±0.3
3.5
3.7
8.6±1.5
3,8
1.4
His
51,4±1.9
4.0
8.0
61.6±9.2
3.8
9.6
23.1±1.1
3.7
3.6
I I.lxl.3
4,9
17
Lys
54,0±1.1
4.2
7.9
105.84-12.9
6.5
15.5
23.4±2.8
3.7
3.4
7.7±1.3
3.4
il
Arg
43,0±2.6
3.3
7.5
60.5±6.8
3.7
10.5
17.0±1.1
2.7
3.0
5.2±0.6
2.3
0.9
13 1.8 08
* nmol/mg - nmol of amino acid per nag of dr 5, HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~2nmol/mg of all amino acids. C~,s, Met, and I y r do not take into account: *** mg/g - mg of amino acid per gram of dry HA sample.
Table 4. Amino acids released after hot acid hydrolysis of podzolic soil HA and fractions "A", "B", and " C + D ' , obtained by tandem SEC-PAGE. HA
Asp
A
B
C+D
l*
2**
3***
1
2
3
1
2
3
1
2
3
142.24-23.3
15.6
18.9
168.8+9.9
14.6
22.5
142.3±40.7
15.6
18.9
48.9+5.0
13.5
6.5
Thr
55.84-10.1
6.1
6.6
73.7+0.6
6.4
8.8
54.8±16.4
6.0
6.5
19.9+1.6
5.5
2,4
Ser
42.9a:7.7
4.7
4.4
59.2+1.7
5.1
6.1
44.7±10.3
4.9
4.6
20.4±2.3
5.7
21
Glu
103.7±19.5
11.4
15.3
135.3:k5.0
11.7
19.9
113.4+30.9
12.4
16.7
49.0+4.2
13.6
7.2
Pro
54.84-9.7
6.0
6.3
66.6+1.6
5.8
7.7
55.8±I 1.5
6. I
6.4
17.3±2.9
4.8
2.(I 4.2
Gly
127.54-21.7
14.0
9.6
161.9±6.5
14.0
12.2
128.8±36.0
14.1
9.7
56.4+6.1
15.7
Ala
87.84-15.8
9.7
7.8
116.84-0.3
10.1
10.4
84.5±25.9
9.2
7,5
31.6±4.2
8.8
Cys
2.94-0.6
0.4
6.0±2.3
0.7
4.7±0.4
0.6
traces
Val
62.74-11.2
9.9
60.9±19. l
7.1
20.6±2.3
Met
5.1±0.7
1.7
6.5±2.5
1.0
2.6±0.9
Ile
37.74-6.7
Leu
50.64-9.0
6.9
7.3
6.7
2,8 traces
7.3
84.64-1.3
0.8
11.6±4.0
5.7
2.4
4.1
4.9
51.1+0.7
4.4
6.7
36.9+10.9
4.0
4.8
13.9±2.0
3.9
1.8
5.6
6.6
72.44-1.2
6.3
9.5
50.0+13.1
5.5
6.6
19.9±1.9
5.5
2.6
04
Tyr
8.1±1.4
1.5
9.5±0.8
1.7
8.6±1.9
1.6
4.6±1.5
Phe
28.14-5.2
3.1
4.6
37.9+1.8
3.3
6.3
28.0±7.4
3.t
4.6
11.1±1.3
3.1
0.8 1.8
His
34.2±6.3
3.8
5.3
45.8±6.0
4.0
7.1
32.8±8.5
3.6
5.1
15.9±1.7
4.4
2.5
Lys
55.5±10.1
6.1
8.1
51.0±3.0
4.4
7.5
53.7±13.0
5.9
7.9
19.84-2.1
5.5
2.9
Arg
25.8±4.7
2.8
4.5
29.6±2.5
2.6
5.2
27.6±6.1
3.0
4.8
10.6±1.7
2.9
1.8
* nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg o f each amino acid)/~nmol/mg of all amino acids, Cys, Met, and I y r do not take into account; *** mg/g - mg of amino acid per gram of dry HA sample.
Amino acid distribution in fractionated HAs
579
Table 5. Amino acids released after hot acid hydrolysis of gray forest soil HA and fractions "A", "B", and "C+D", obtained by tandem SEC-PAGE. HA 1"
2**
A 3***
1
B 2
3
1
C+D 2
3
1
2
3
Asp 64.0-J:17.8 13.9 8.5 111.2:t:61.6 12.4 14.8 93.2-4-28.0 14.6 12.4 29.9-±2.4 18.5 4.0 Thr 23.7±7.5 5.2 2.8 57.14-32.4 6.4 6.8 35.44-10.5 5.6 4.2 7.2±0.6 4.5 0.9 Ser 22.14-7.6 4.8 2.3 51.44-26.8 5.7 5.3 36.34-9.1 5.7 3.7 9.14-1.4 5.6 0.9 Glu 50.84-13.4 11.0 7.5 94.74-49.2 10.6 13.9 78.4±22.0 12.3 11.5 16.9-±1.6 10.5 2.5 Pro 33.84-7.8 7.5 3.9 45.4±22.5 5.1 5.2 34.6±12.3 5.4 4.0 7.0-±0.5 4.3 0.8 Gly 59.94-17.1 13.0 4.5 120.7±66.5 13.5 9.1 92.9-±25.1 14.6 7.0 26.1±2.9 16.2 2.0 Ala 38.64-11.0 8.4 3.8 91.7±50.2 10.2 9.1 59.8±16.5 9.4 5.9 12.2±1.8 7.5 1.2 Cys 2.34-0.6 0.3 6.3±3.2 0.8 4.6±1.0 0.6 traces traces Val 25.9-a:15.3 5.6 3.0 61.5±33.5 6.9 7.2 35.54-10.3 5.6 4.2 7.4±0.8 4.6 0.9 Met 2.44-0.6 0.4 9.7±7.6 1.4 8.7±3.5 1.3 1.94-0.2 0,3 Ile 19.04-2.1 4.1 2.5 34.94-25.5 3.9 4.6 18.3±7.6 2.9 2.4 4.8±0.2 3.0 0.6 Leu 29.84-3.6 6.5 3.9 58.8±42.4 6.6 7.7 27.8±11.6 4.4 3.6 7.94-0.1 4.9 1.0 Tyr 5.04-2.0 0.9 9.64-5.6 1.7 4.4±0.5 0.8 1.8±0.1 0.3 Phe 15.54-4.6 3.4 2.6 30.6±14.9 3.4 5.1 17.24-5.2 2.7 2.8 4.5±0.2 2.8 0.7 His 27.74-10.3 6.0 4.3 49.54-26.8 5.5 7.7 37.54-9.9 5.9 5.8 8.0±1.2 5.0 1.2 Lys 31.04-7.9 6.7 4.5 43.3±1.4 4.8 6.3 42.24-13.2 6.6 6.2 13.8±0.8 8.5 2.0 Arg 18.1±4.8 3.9 3.2 44.6±5.7 5.0 7.8 28.7±7.1 4.5 5.0 6.8±2.0 4.2 1.2 * nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~nmoi/mg of all amino acids, Cys, Met, and Tyr do not take into account; *** mg/g - mg of amino acid per gram of dry HA sample.
Table 6. Amino acids released after hot acid hydrolysis of chemozem HA and fractions "A", "B", and "C+D", obtained by tandem SECPAGE. HA 1'
2**
A 3***
1
B 2
3
1
Asp 81.7+11.3 17.0 10.9 187.94-49.9 18.1 25.0 118.6+8.0 Thr 22.7±3.3 4.7 2.7 52.8±8.5 5.1 6.3 29.9±0.3 Ser 20.9±3.1 4.4 2.2 47.8±4.2 4.6 4.9 29.6±4.2 Glu 55.5+7.2 11.6 8.2 121.2±16.1 11.6 17.8 81.1±16.0 Pro 33.4+3.5 7.0 3.8 47.5±2.0 4.6 5.5 35.0±6.2 Gly 65.4+8.8 13.6 4.9 145.8±33.1 14.0 10.9 90.4±5.7 Ala 39.4±6.3 8.2 3.9 102.24-26.3 9.8 10.1 54.4±1.3 Cys 2.2±0.6 0.3 10.74-2.2 1.3 5.54-0.5 Val 27.6±3.9 5.8 3.2 69.4+15.6 6.7 8.1 36.0~1.2 Met 2.14-0.2 0.3 7.94-5.5 1.2 5.24-2.9 lie 19.14-1.9 4.0 2.5 40.9±6.6 3.9 5.4 18.54-1.8 Leu 28.2±2.9 5.9 3.7 66.04-13.1 6.3 8.7 27.5±2.5 Tyr 4.8±0.5 0.9 7.1±2.1 1.3 3.94-1.7 Phe 14.6a:2.0 3.0 2.4 30.54-3.4 2.9 5.0 16.04-2.8 His 26.0-±5.1 5.4 4.0 44.1±8.1 4.2 6.8 28.1±0.3 Lys 28.1±3.5 5.9 4.1 47.3±1.6 4.5 6.9 35.8±5.4 Arg 16.84-2.0 3.5 2.9 37.34-9.5 3.6 6.5 22.0-±8.7 * nmol/mg - nmol of amino acid per mg of dry HA sample; ** n m o l % - 100(nmol/mg of each amino acid)/~nmol/mg of all amino acids, Cys, *** mg/g - m g of amino acid per gram of dry HA sample.
C+D 2
3
1
2
3
19.0 4.8 4.8 13.0 5.6 14.5 8.7
15.8 3.6 3.1 11.9 4.0 6.8 5.4 0.7 4.2 0.8 2.4 3.6 0.7 2,6 4.4 5.2 3.8
31.4±3.8 8.1±0.3 9.4±2.1 21.1+3.3 10.5±1.8 30.7±3.2 14.74-0.1 traces 9.34-0.1 1.7±0.3 5.0-±0.3 7.94-1.2 1.5±0.7 5.6±0.4 9.5±0.8 11.4±2.5 9.4±3.9
17.1 4.4 5.1 11.5 5.7 16.7 8.0
4.2 1.0 1.0 3.1 1.2 2.3 1.5 traces 1.1 0.3 0.7 1.0 0.3 0.9 1.5 1.7 1.6
5.8 3.0 4.4 2.6 4.5 5.7 3.5
5.1 2.7 4.3 3.0 5.2 6.2 5.1
Met, and Tyr do not take into account;
580
O.E. Trubetskaya et al.
Table 7. Amino acids released after hot acid hydrolysis of red soil HA and fractions " A ' , "B", and "C+D", obtained by tandem SEC-PAGE. HA 1'
A
B
C+D
2**
3***
1
2
3
I
2
3
I
2
Asp 82.2+4.0 Thr 39.6+1.3 Ser 35.8+3.3
13.0
10.9
140.0+15.5
13.8
18.6
126.9+20.9
14.4
16.9
68.8+26.6
15.4
9.2
6.2
4.7
68.6+6.3
6.8
8.2
55.1±8.9
6.2
6.6
22.8+8.1
5.1
2.7
5.6
3.7
66.8+6.6
6.6
6.9
52.0+7.3
5.9
5.4
25.3±6.8
5.6
2.~
Glu
66.5+3.6
10.5
9.8
104.2+12.4
10.3
15.3
106.6±16.8
12.1
15.7
48.6+12.6
10.8
7.2
Pro
53.7+5.9
8.5
6.2
62.1+10.5
6.1
7.1
60.8+6 8
6.9
7,0
5.9
3.(i
Gly
79.5+6.3
12.5
6.0
141.8+14.2
14.0
10.6
127.9+20.2
14.5
9.6
78.8+23.8
17.6
5.9
Ala
53.9+3.0
8.5
5.3
102.4+8.8
10.1
10.1
82.9+14.6
9.4
8.2
38.7+14.2
8.6
3.8
Cys 2.1±0.1 Val 40.8±0.4 Met 1.9+0.9
0.3
6.9+t:3.7
0.8
6.3±1.6
6.4
4.8
70.2+7.4
6.9
8.2
54.2±6.8
0.3
7.0~1.6
-
1.0
5.9±22
Ile
26.1+2.2
4.1
3.4
35.7+4.7
3.5
4.7
30.0±3.4
3.4
3.9
15.8+3.5
3.5
Leu 40.7±4.5
6.4
5.3
56.0+8,6
5.5
7.3
45.8+4.0
5.2
6.0
24.5+4.7
5.5
1.4
11.7:~2.0
2.1
8.3±2.1
1,5
5.1±1.6
Tyr
8.0+1.1
Phe 26.2±1.3 His 39.1+5.0
6.1
26.3t7.9
0.8
traces
6.3
23.9+7.7
0.9
1.9+0.7
traces 5.3
2.8 0.3 2.1 3.2 0.9
4.1
4.3
40.6:L9.0
4.0
6.7
28.4±3.8
3.2
4.7
14.8+3.8
3.3
2.4
6.2
6.1
61.7+10.4
6.1
9.6
46.6±6.0
5.3
7.2
23.5±5.9
5.2
3.6
30.4+1.4
4.8
4.4
38.4+4.2
3.8
5.6
41.4+5.6
4.7
6.1
20.5±3.8
4.6
3.{)
Arg 20.2+1.9
3.2
3.5
26.3±2.8
2.6
4.6
25.4±8.5
2.9
4.4
15.7+2.7
3.5
2.7
Lys
* nmol/mg - nmol of amino acid per mg of dry HA sample; ** nmol% - 100(nmol/mg of each amino acid)/~nmol/mg of all amino acids, Cys, Met, and Tyr do not take into account: *** mg/g - m g of amino acid per gram of dry HA sample.
Table 8. Amino acids weight content (%) in five soil HAs of different origin and fractions "'A", "B", and "C+D", obtained by SEC-PAGE.
Name of the soil Arctic soil Podzolic soil Gray forest soil Chernozem Red soil
HA
A
B
C+D
16.3
21.7
7.8
2.8
11.3
13.9
I 1.5
4.4
5.9
11,5
8. I
2.1
6.1
13,2
7.9
2.3
8.0
12,7
11.1
5.5
soils investigated contained from 7.5 to 11.5% of amino acids. The smallest amino acids content, from 2.1 to 5.5%, was found in low MS fractions "C+D". The larger content of amino acids from high MS fractions may be due to the hydrolysis of peptide of relatively greater chain length.
CONCLUSION
The five HAs from soils of different genesis and climatic conditions were fractionated by coupling SECPAGE. Preparative quantities of three highly purified
HA fractions ("A", "B", and "C+D") with defined molecular and electrophoretic entities from each soil HA sample were obtained. Amino acids contents of each HA and its fractions were studied. The highest amino acids content (11.5-21.7%) was found in high MS fractions "A" and the smallest amino acids content (2.1-5.5%) was found in low MS fractions "C+D". If the structural complexity of humic macromolecules is taken into account, a separation of HA into less complex fractions with defined molecular and electrophoretic entitles, which differ by their content of amino acids, could be of great value for studies of physicalchemical structures of HA and its genesis in soil.
Amino acid distribution in fractionated HAS
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