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Importance of the molecular weight of humic and fulvic acids in determining their effects on protease activity
Soil Biol. Biochem. Vol. 3 pp. 249-257. P e r g a m o n Press 1971. Printed in Great Britain
IMPORTANCE OF THE MOLECULAR WEIGHT OF H U M I C A N D FULVIC ACIDS 1N D E T E R M I N I N G THEIR EFFECTS ON PROTEASE ACTIVITY J. H. A. BUTLER and J. N. LADD Division of Soils, CSIRO, Glen Osmond, South Australia
(Accepted 28 September 1970) Summary--Humic and fulvic acids from three soils were tested for their effects on the activities of four proteolytic enzymes. Humic acids behaved similarly and, as a group, had a greater effect on enzyme activity than fulvic acids. This difference was not related to the carboxyl content of the preparations but may be due in part to differences in their molecular weights. Thus, using membrane filtration, the humic acid from a podzol B horizon was shown to contain a greater proportion of high molecular weight components than did the corresponding fulvic acid; and high molecular weight fractions of both humic and fulvic acids had greater effects on enzyme activity than the respective, low molecular weight fractions. This indicates that molecular weight is of overriding importance in determining the magnitude of the effect on enzyme activity. The possibility is discussed that the effects on enzyme activity may also reflect differences in the structure and rigidity of humic and fulvic acid molecules. INTRODUCTION
NEUTRALIZED solutions of soil humic acids markedly affect protease activities (Ladd, Brisbane and Butler, 1968; Ladd and Butler, 1969a, b) probably by binding the enzymes at cation exchange sites on the humic acid polymer (Butler and Ladd, 1969b; Ladd and Butler 1970). The magnitude of the effect of a humic acid, methylated to different extents, was directly related to its free carboxyl content (Butler and Ladd, 1969b). However, humic acids of different nominal molecular weight ranges, obtained using Sephadex gels, affected enzyme activities (trypsin, papain) to an extent inversely related to the carboxyl content of the humic fractions (Ladd and Butler, 1969b). The relative importance of carboxyl content and molecular size has been studied further using humic and fulvic acid preparations. It was anticipated from other studies (Kononova, 1966--p. 88; Schnitzer and Desjardins, 1962; Felbeck, 1965) that fulvic acids would have lower average molecular weights and higher carboxyl contents than humic acids from the same soil, but that the basic structure of fulvic and humic acids would be very similar. MATERIALS AND METHODS
Soils Replicate samples of soil were collected from three locations in South Australia, soil 22 from the B horizon of a ground water podzol near Wickham Hill, soil 24 from 0 - 7 . 5 cm of the Waite Agricultural Research Institute experimental plots, C.1. permanent pasture, and soil 25 from 0-23 cm of a podzolized sand under pasture near Mt Compass. Soil 22 was classified by Northcote (1965) as Uc 2.33, soil 24 as Dr 2.23 and soil 25 as Uc 2.21.
Extraction of humic and fulvic acids The soils were dried at 35°C, ground, sieved (1.0 mm) and samples (soil 22, 17 kg; soils 24 and 25, 0.5 kg) were extracted with 0-5N N a O H (Soil 22, 20 1.; soils 24 and 25, 249
250
J.H.A. BUTLER AND J. N. LADD
1 •5 1.) under N2 for 18 hr on a roller at room temperature. After settling, each supernatant was centrifuged at 10,000 g for 60 min to remove clay, and concentrated to one-fifth its original volume, either on a cyclone evaporator at 35°C (22C)* or freeze drier (24C and 25C). Each extract was dialysed against distilled water until its salt content was low. Humic acid was precipitated by adding 5N HC1 with vigorous stirring to p H 1.5, and recovered by centrifugation. The moist humic acid was dissolved in phosphate buffer at pH 7, centrifuged at 10,000g for 2 hr to remove clay and then precipitated by acid to pH 1 •5. This process was repeated twice. After the final centrifugation at p H 7, the supernatant was dialysed twice against 100 volumes of 0.1N HC1 and then against distilled water until chloride-free. The resulting suspension of humic acid was freeze dried and stored in a desiccator. Each supernatant remaining after the initial precipitation of humic acid was concentrated where necessary by cyclone evaporation to about 200 ml, centrifuged at 10,000 g for 3 hr to remove clay, and passed through a Dowez 50 × 8 resin column, H + form (7 × 50 cm for 22CF solution and 4 × 25 cm for 24CF and 25CF solutions). The fulvic acid solutions were dialysed twice against 0. l y HCI, then against distilled water until chloride-free, freeze dried and stored in a desiccator.
Fractionation of humie and fulvie acids (a) Membrane filtration. Samples of 22CH and 22CF were each separated into three fractions of nominal molecular weights 1000-10,000, 10,000-30,000 and greater than 30,000 by membrane filtration as follows. One litre of solutions containing 1 g of either 22CH or 22CF in 0.1M NaCl at p H 7-8 was added to a stirred 1 1. pressure filtration vessel~ fitted with a 3-in. dia. Diaflo PM30 membrane'~ (nominal molecular weight cut-off, 30,000). The cell was pressurized to 6.3 kg/cm 2 through a reservoir containing 1 1. of 0.1M NaC1 which thus replaced solution passing out of the cell. Filtration was continued until 1900 ml of effluent were collected. This effluent was then filtered through a Diaflo P M I 0 membrane (nominal molecular weight cut-off, 10,000), washed through with 500 ml of 0.1M NaCI, and filtration continued until 100 ml of solution remained in the cell. The effluent which had passed through the PM10 membrane was then filtered through a Diaflo UM2 membrane (nominal molecular weight cut-off, 1,000) and washed through with 250 ml 0- 1M NaCl, filtration being continued until 100 ml of solution remained in the cell. The pH of the solutions retained by the PM30, PM10 and UM 2 membranes was adjusted to 2 with 6N HC1. The precipitated humic acids were recovered by centrifugation and the fulvic acid solutions were desalted by washing with water on a Diaflo UM 2 membrane and freeze dried. (b) Gel filtration. Humic and fulvic acids from soil 22 were fractionated by Sephadex gel filtration (small scale) as described by Butler and Ladd (1969a). (c) Gel eleetrophoresis. Preparations 22CH, 22CF and each of their Diaflo fractions were further studied using polyacrylamide gel electrophoresis. Samples (2.5 mg and 7.5 mg for humic and fulvic acids, respectively) were dissolved in 0.5 ml. Tris buffer, pH 8.3 containing 2 0 ~ sucrose. Aliquots (20 ~1) were applied to the top of 30~ gel rods (Davis, 1964) in a Canalco electrophoresis apparatus.+ A current of 4 mA per gel rod (4.0 × 0.5 cm) was applied for 30 rain. The gel rod was extruded and two sections were cut out, one from 1 mm * 22C, 24C, 25C are alkaline extracts from soils 22, 24 and 25 respectively; F and H designate fulvic and humic acids respectively. t Available from the Amicon Corp. Lexington, Massachusetts, U.S.A. ++Available from Canalco, Bethesda, Maryland, U.S.A.
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
251
in front of the salt front to 3 mm behind, and the other, the remainder of the gel rod back to the origin. These sections were then placed in separate centrifuge tubes, the gel crushed with a glass rod, allowed to stand overnight in distilled water and centrifuged. The absorbance at 400 nm and the volumes of the extracts were measured and, after correction for material extracted from a blank section of gel, the percentage distribution of fast moving material was determined.
Properties of humic and fulvic acids Carboxyl contents of the preparation were determined by methylation followed by hydrolysis (Butler and Ladd, 1969a). Ash contents were determined after heating samples in platinum boats for 4 hr at 600°C, carbohydrate contents by the method described by Oades (1967), and amino-acid release by acid hydrolysis and absorbance spectra as previously described (Butler and Ladd, 1969a).
Enzyme substrates and assay procedures The sources of enzymes and substrates* have been described (Ladd and Butler, 1969a, b). Table 1 summarizes protease assay conditions. Fulvic and humic acids were dissolved in 0"025M Tris buffer, pH 7.5 and preincubated with buffered enzyme for either 30 min (carboxypeptidase) or 60 min (other proteases) at the specified temperature. Substrate was TABLE1. ASSAYSOFPROTEASEACTIVITY Enzyme (mg)
Substrate (~.mol)
Pronase (0.1) Trypsin (0.06) Carboxypeptidase (0.005) Papain (0"008)
Z-gly-leu (1.0) BAA (1.0) Z-gly-phe (1.0) BAA (1"0)
Buffer (/~mol) Tris (20) Tris (20) Tris (20) Citrate (40), EDTA (1), mercaptoethanol (10)
Temp. (°C)
pH
Reaction time (rain)
37
7- 5
70
25
7"5
60
25
7.5
30
37
5" 5
60
Total reaction volume: 1 ml added and after the specified reaction time, enzyme activity was stopped with 0.1 ml 5N HCI. Ammonia or amino acid released by enzyme action was determined with a ninhydrin reagent (Moore and Stein, 1954). Values were corrected for those of controls without substrate. Activities in the presence of humic or fulvic acids were expressed as percentages of those of corresponding reaction mixtures without humic compounds. RESULTS
Properties of humic and fulvic acids Figure 1 shows the effect of varying concentrations of three humic acids and three fulvic acids on the activities of four proteases. For each enzyme assay, composite graphs illustrate * Substrate abbreviations: N-benzoyl-L-arginineamide (BAA); N-carbobenzoxy-glycylleucine (Z-gly-leu); N-carbobenzoxy-glycolphenylalanine (Z-gly-phe).
252
J . H . A . BUTLER AND J. N. L A D D
t h e results o f t h r e e s e p a r a t e e x p e r i m e n t s , e a c h o f w h i c h tested t h e effect o f the h u m i c a n d fulvic acids e x t r a c t e d f r o m the s a m e soil. H u m i c a c i d s f r o m different soils b e h a v e d similarly, a n d as a g r o u p , differed f r o m the fulvic acids. H u m i c acids i n h i b i t e d trypsin, p r o n a s e a n d c a r b o x y p e p t i d a s e a n d s t i m u l a t e d 120 IOO
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CONCENTRATION OF HUMIC AND FUL.VlC ACIDS, ~g/ml
Fro. 1. The effect of different levels of humic and fulvic acids on the activity of four proteolytie enzymes. Conditions of assay as in Table 1. Enzymes preincubated for 1 hr (0.5 hr with carboxypeptidase) with the humic or fulvic acids before addition of substrate. Reference enzyme activities measured without humic preparations. A 22CH • 22CF [] 24CH • 24CF O 25CH • 25CF TABLE 2. Preparation
PROPERTIES OF HUMIC AND FULVIC ACIDS
Ash (~)
Carbohydrate (%)
Amino acid* (%)
Absorbancet 260 nm 450 nm
22CF 24CF 25CF
10.2 4"2 5-5
2"3 28"2 29-3
2.1 9-3 7.4
23-8 6.4 7.9
22CH 24CH 25CH
1 "0 4"4 7"0
0" 8 4-4 2.7
6.6 12-3 10.6
40.1 27.5 25.6
Carboxyl Uncorr.
m-equiv/g Corr.++
1.60 0.44 0-62
5.5 2-9 2.7
6.4 5.0 4.7
5"95 4-02 3"28
4"4 3.6 3-3
4-8 4.5 4" I
* Percentage amino acid-N released by acid hydrolysis x 6" 25. t Absorbance of solution at 1 mg/ml, pH 7.0. Expressed on an ash-free, carbohydrate-free and amino acid-free basis.
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
253
p a p a i n . F u l v i c acids inhibited trypsin a n d p r o n a s e less t h a n d i d h u m i c acids, h a d little o r no effect on p a p a i n a n d slightly s t i m u l a t e d c a r b o x y p e p t i d a s e . T a b l e 2 shows t h a t several p r o p e r t i e s o f the p r e p a r a t i o n s overlap. C a r b o h y d r a t e a n d acidh y d r o l y s a b l e a m i n o acid contents o f h u m i c a n d fulvic acids f r o m the p o d z o l B h o r i z o n were lower t h a n those o f the c o r r e s p o n d i n g p r e p a r a t i o n s f r o m the t o p soils. F u l v i c acid c o n t a i n e d m o r e c a r b o h y d r a t e a n d less a c i d - h y d r o l y s a b l e a m i n o acid t h a n h u m i c acid f r o m the same soil. F u l v i c acids also were o f higher c a r b o x y l c o n t e n t when expressed on an ash-, c a r b o h y d r a t e - , a n d a m i n o acid-free basis. The effects on enzyme activity o f h u m i c a n d fulvic acids, either within a n d especially between groups, correlates neither with their t o t a l c a r b o x y l c o n t e n t n o r with their contents o f i n o r g a n i c material, c a r b o h y d r a t e a n d acidh y d r o l y s a b l e a m i n o acids.
Fractionation of humic and fulvic acids F r a c t i o n a t i o n o f p o d z o l B h u m i c acid (22CH) a n d fulvic acid (22CF) b y Diaflo ultrafiltration, using three m e m b r a n e s o f different exclusion limits, showed t h a t m o s t o f the TABLE 3. NOMINALMOLECULARWEIGHTDISTRIBUTIONOF A HUMICAND A FULVICACID Preparation
Diaflo ultrafiltration Per cent by weight of nominal molecular weight* 1000-10,000
22CF 22CH
10,000-30,000
78 10
> 30,000
10 4
12 86
Percent by absorbance(260 nrn) 22CF 22CH
75.3 11.3
10.4 5.3
14- 3 83.4
Sephadex gel filtration Per cent by absorbance (260 nm) of nominal molecular weight* < 5000 22CF 22CH
25 40
< 10,000
< 50,000
44 54
55 56
* Nominal values based on manufacturers calibrations. TABLE4.
PER CENT LOW MOLECULAR WEIGHT MATERIAL IN HUMIC PREPARATIONS BY ACRYLAMIDE GEL ELECTROPHORESIS
Preparation 22CF 22CH 22CF 1000-10,000:~ > 30,000 22CH 1000-10,000,* > 30,000
Low molecular weight1" ( ~ )
Recovery (~o)
66 35 72 21 61 24
110 92 93 109 96 98
I" Material travelling with salt front. ~. Fractions obtained by Diaflo ultrafiltration. Percentages calculated from absorbances at 400 nm.
254
J.H.A.
B U T L E R A N D J. N. L A D D
fulvic acid distributed in the nominal molecular weight range 1000-I0,000, whereas most of the humic acid components were of nominal molecular weight greater than 30,000 (Table 3). The results obtained by polyacrylamide gel electrophoresis of humic acid 22CH and fulvic acid 22CF and their Diaflo fractions are shown in Table 4. The fractionation obtained using the small pore polyacrylamide gels (30~ w/v) reflected differences in molecular size rather than differences in charge as there was no separation in the large pore gel (7~ w/v) where the preparations ran with the salt front. Fractionation by Sephadex gel filtration indicated that the humic acid had a higher proportion of low molecular weight components than the fulvic acid (Table 3).
Properties of humic andfuh;ic acidfractions All properties described are of fractions obtained by Diaflo ultrafiltration of humic acid 22CH and fulvic acid 22CF. Humic acid components of increasing nominal molecular weight were increasingly effective as inhibitors of trypsin, pronase and carboxypeptidase activities. Stimulation of papain activity by humic acid 22CH was due solely to its components of relatively high molecular weight (Fig. 2). I00
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FIG.. 2.
The effect of different molecular weight fractions of 2 2 C H and 2 2 C F on the activity of four proteolytic enzymes. Conditions of assay as in Table 1 and Fig. I. O [] A
22CH 1,000-10,000 2 2 C H 10,000-30,000 22CH >30,000
• • •
2 2 C F 1,000-10,000 2 2 C F 10,000-30,000 22CF >30,000
The fulvic acid fraction of highest molecular weight had little or no effect on papain activity. Fulvic acid components of increasing nominal molecular weight were in general increasingly effective inhibitors of trypsin and pronase, although slight stimulation of
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
255
pronase activities by low concentrations of fulvic acids was obtained. The lowest molecular weight fulvic acid components also stimulated carboxypeptidase activity slightly throughout their concentration range, whereas inhibition was obtained with the other fractions and was related both to their concentration and their molecular weight. The results shown in Fig. 2 also show that in general humic acids of a given molecular weight range have significantly more effect on enzyme activity than fulvic acids of the same molecular weight range. Other properties of the fractions are shown in Table 5. The effects on enzyme activity of the various fractions were not directly correlated with their carboxyl content. TABLE 5. PROPERTIES OF HUMIC AND FULVIC ACID FRACTIONS OBTAINED BY DIAFLO ULTRAFILTRATION
Preparation
Ash (~)
Carbohydrate (%)
Amino acid* (%)
Absorbance'~ 260 nm 450 nm
22CF 1000-10,000 10,000-30,000 > 30,000
23"1 11.1 5.3
1-8 -3-4
2"0 2-0 2"6
22-6 25.2 26"8
22CH 1000-10,000 10,000-30,000 > 30,000
4-2 -4"9
0.2 0.6 0"8
4"6 6"4 7"4
42.8 43-6 36"0
Carboxyl Uncorr.
m-equiv/g Corr.++
1"66 1"95 2"74
3"6 4-1 4"6
4"9 -5.2
6-02 6"80 6"10
4"4 4"1 3"9
4"8 -4"5
* Per cent amino acid-N released by acid hydrolysis × 6.25. t Absorbance of solution of concentration 1 mg/ml, pH 7"0. **Corrected to an ash-, carbohydrate- and amino acid-free basis.
DISCUSSION
The molecular weight distribution of either a humic or a fulvic acid preparation differed according to the technique used. The molecular weights shown (Table 3) are derived entirely from the manufacturers calibrations of Diaflo membranes and Sephadex gels, and in the case of acrylamide gel electrophoresis no calibration was available. Thus molecular weights quoted are nominal and it is not surprising that the values do not agree with many of the widely varying values quoted in the literature (Mehta et al., 1963; Schnitzer and Skinner, 1968; Kononova, 1966). However comparisons between fractions obtained by a given method are valid. Based on absorbance (260 nm) of fractionated material, 54~o of humic acid and 447o of fulvic acid were of molecular weight less than 10,000 when fractionated by Sephadex gel filtration, whereas using Diaflo membrane filtration 119/o of the humic acid and 7 5 ~ of the fulvic acid were of molecular weights 1000-10,000 (Table 3). It is obvious that other factors, e.g. adsorption and exclusion of charged molecules, may be influencing the separations obtained by one or both of the methods. However, the results obtained by membrane filtration are considered to be the more correct. Thus, by this technique fulvic acid 22CF was of lower molecular weight than humic acid 22CH, which is consistent with the results obtained by polyacrylamide gel electrophoresis (Table 4) and which agrees with the general conclusions of other workers (Scheffer and Ulrich, 1960--p. 41; Schnitzer and Desjardins, 1962).
256
J.H.A. BUTLER AND J. N. LADD
The evidence suggests that the Sephadex gel filtration method leads to anomalously high values for the molecular weights of the fulvic acid. Schnitzer and Skinner (1968) also consider that values of molecular weight obtained by fractionation of a podzol B fulvic acid by Sephadex gel filtration were two to ten times higher than those obtained by other methods. Although quantitatively the results of Sephadex gel fractionation of humic acids did not agree well with the Diaflo fractionation figures, acrylamide gel electrophoresis confirmed that humic acid fractions previously obtained with Sephadex (Butler and Ladd, 1969a) graded in molecular weight as expected (Butler, unpublished). Also, the nominated higher molecular weight components of humic acids fractionated by either technique were the more effective stimulators of papain activity and inhibitors of trypsin activity (Ladd and Butler, 1969b--Fig. 2). Despite similar carboxyl contents of all preparations, humic acids have a greater effect on enzyme activity than fulvic acids (Fig. 1) and high molecular weight components of both humic and fulvic acids have a greater effect than the respective low molecular weight components (Fig. 2). In previous papers, Ladd and Butler (1969a, 1970) suggest that changes in enzyme activity with humic acids may result from changes in enzyme conformation due to the enzyme being bound to the humic polymer by a cation exchange mechanism. The extent to which an enzyme molecule is distorted by such a mechanism would be determined in part by the rigidity, size, shape and charge density of the humic molecules. Thus a humic molecule of high molecular weight, because of its inherent rigidity in comparison with an equal weight of smaller molecules should distort the enzyme further. The direct relationship between molecular weight of humic compounds and their effect on enzyme activity is consistent with the effects on proteases of humic and fulvic acids considering their respective molecular weight distributions. However, Fig. 2 shows that humic and fulvic acid fractions of the same molecular weight range differ in their effects on enzymes, and demonstrates that other factors additional to differences in molecular weight distribution are important. Qualitative differences between humic and fulvic acids cannot be stated with certainty because their structure, including the distribution of the carboxyl groups is little known. However, the humic acid polymer is considered to be more aromatic and more condensed than the fulvic acid polymer (Scheffer and Ulrich, 1960--p. 41; Kononova, 1966--p. 91). Hence humic acids should be more rigid than fulvic acids and more able to distort enzyme molecules bound to them. None of the effects on enzymes are related to the ash, amino acid or carbohydrate contents of the humic preparations. This is shown well in the carboxypeptidase assay where all the fulvic acids exert essentially the same effect, yet their ash contents range from 4.2 to 10.2%, their carbohydrate contents range from 2-3 to 29-3% and amino acid-N yields vary from 0- 34 to 1.49%. Acknowledgement--The
authors are indebted to Mr M. AMATOfor technical assistance.
REFERENCES
BUTLERJ. H. A. and LADDJ. N. (1969a) Effect of extractant and molecular size on the optical and chemical properties of soil humic acids. Aust. J. Soil Res. 7, 229-239. BUTLERJ. H. A. and LADDJ. N. (1969b) The effect of methylation of humic acids on their influence on proteolytic enzyme activity. Aust. J. Soil Res. 7, 263-268. DAVISB. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. N . Y . Acad. Sci. 121, 404-427. FELBECKG. T. (1965) Structural chemistry of soil humic substances. Adv. Agron. 17, 327-368.
I M P O R T A N C E OF THE M O L E C U L A R WEIGHT OF H U M I C A N D FULVIC ACIDS
257
KONONOVA M. M. (1966) Soil Organic Matter, 2nd English edn, Pergamon Press, Oxford. LADD J. N., BRISBANEP. G. and BUTLERJ. H. A. (1968) The Susceptibility of Nitrogenous Components of Humic Acids to Enzyme Attack: Inhibition of 'Pronase' Activity, Transactions of the Ninth Congress of the International Society for Soil Science, Adelaide, Vol. 3, pp. 319-327. LADD J. N. and BUTLER H. J. A. (1969a) Inhibitory effect of soil humic compounds on the proteolytic enzyme 'Pronase'. Aust. J. Soil Res. 7, 241-251. LADD J. N. and BUTLERJ. H. A. (1969b) Inhibition and stimulation of proteolytic enzyme activities by soil humic acids. Aust. J. Soil Res. 7, 253-261. LADD J. N. and BUTLERJ. H. A. (1970) The effect of inorganic cations on the inhibition and stimulation of protease activity by soil humic acids. Soil Biol. Biochem. 2, 1-8. MEHTA N. C. DUBACH P. and DEUEL H. (1963) Untersuchtmgen uber die Molekularge wichts-Verteilung von Huminstoffen dutch Gelfiltration an Sephadex. Z. PflErndhr. Dang. Bodenk. 102 (2), 128-137. MOORE S. and STEIN W. H. (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. biol. Chem. 211,907-911. NORTHCOTEK. H. (1965) A Factual Key for the Recognition of Australian Soils, CSIRO Australian Division of Soils Divisional Report No. 2/65. OADESJ. M. (1967) Carbohydrates in some Australian soils. Aust. J. Soil Res. 5, 103-116. SCHEFFERF. and ULRICH B. (1960) Humus und Humusdungung, Ferdinand Enke, Stuttgart. SCHNITZER M. and DESJARDINSJ. G. (1962) Molecular and equivalent weights of the organic matter of a podzol. Proc. Soil Sci. Soc. Am. 26, 362-365. SCI-INITZER M. and SKINNER S. I. M. (1968) Gel Filtration of Fulvic Acid, a Soil Humic Compound. In Isotopes and Radiation in Soil Organic-Matter Studies, pp. 41-55, IAEA, Vienna.
sor~ 3/3---0
IMPORTANCE OF THE MOLECULAR WEIGHT OF H U M I C A N D FULVIC ACIDS 1N D E T E R M I N I N G THEIR EFFECTS ON PROTEASE ACTIVITY J. H. A. BUTLER and J. N. LADD Division of Soils, CSIRO, Glen Osmond, South Australia
(Accepted 28 September 1970) Summary--Humic and fulvic acids from three soils were tested for their effects on the activities of four proteolytic enzymes. Humic acids behaved similarly and, as a group, had a greater effect on enzyme activity than fulvic acids. This difference was not related to the carboxyl content of the preparations but may be due in part to differences in their molecular weights. Thus, using membrane filtration, the humic acid from a podzol B horizon was shown to contain a greater proportion of high molecular weight components than did the corresponding fulvic acid; and high molecular weight fractions of both humic and fulvic acids had greater effects on enzyme activity than the respective, low molecular weight fractions. This indicates that molecular weight is of overriding importance in determining the magnitude of the effect on enzyme activity. The possibility is discussed that the effects on enzyme activity may also reflect differences in the structure and rigidity of humic and fulvic acid molecules. INTRODUCTION
NEUTRALIZED solutions of soil humic acids markedly affect protease activities (Ladd, Brisbane and Butler, 1968; Ladd and Butler, 1969a, b) probably by binding the enzymes at cation exchange sites on the humic acid polymer (Butler and Ladd, 1969b; Ladd and Butler 1970). The magnitude of the effect of a humic acid, methylated to different extents, was directly related to its free carboxyl content (Butler and Ladd, 1969b). However, humic acids of different nominal molecular weight ranges, obtained using Sephadex gels, affected enzyme activities (trypsin, papain) to an extent inversely related to the carboxyl content of the humic fractions (Ladd and Butler, 1969b). The relative importance of carboxyl content and molecular size has been studied further using humic and fulvic acid preparations. It was anticipated from other studies (Kononova, 1966--p. 88; Schnitzer and Desjardins, 1962; Felbeck, 1965) that fulvic acids would have lower average molecular weights and higher carboxyl contents than humic acids from the same soil, but that the basic structure of fulvic and humic acids would be very similar. MATERIALS AND METHODS
Soils Replicate samples of soil were collected from three locations in South Australia, soil 22 from the B horizon of a ground water podzol near Wickham Hill, soil 24 from 0 - 7 . 5 cm of the Waite Agricultural Research Institute experimental plots, C.1. permanent pasture, and soil 25 from 0-23 cm of a podzolized sand under pasture near Mt Compass. Soil 22 was classified by Northcote (1965) as Uc 2.33, soil 24 as Dr 2.23 and soil 25 as Uc 2.21.
Extraction of humic and fulvic acids The soils were dried at 35°C, ground, sieved (1.0 mm) and samples (soil 22, 17 kg; soils 24 and 25, 0.5 kg) were extracted with 0-5N N a O H (Soil 22, 20 1.; soils 24 and 25, 249
250
J.H.A. BUTLER AND J. N. LADD
1 •5 1.) under N2 for 18 hr on a roller at room temperature. After settling, each supernatant was centrifuged at 10,000 g for 60 min to remove clay, and concentrated to one-fifth its original volume, either on a cyclone evaporator at 35°C (22C)* or freeze drier (24C and 25C). Each extract was dialysed against distilled water until its salt content was low. Humic acid was precipitated by adding 5N HC1 with vigorous stirring to p H 1.5, and recovered by centrifugation. The moist humic acid was dissolved in phosphate buffer at pH 7, centrifuged at 10,000g for 2 hr to remove clay and then precipitated by acid to pH 1 •5. This process was repeated twice. After the final centrifugation at p H 7, the supernatant was dialysed twice against 100 volumes of 0.1N HC1 and then against distilled water until chloride-free. The resulting suspension of humic acid was freeze dried and stored in a desiccator. Each supernatant remaining after the initial precipitation of humic acid was concentrated where necessary by cyclone evaporation to about 200 ml, centrifuged at 10,000 g for 3 hr to remove clay, and passed through a Dowez 50 × 8 resin column, H + form (7 × 50 cm for 22CF solution and 4 × 25 cm for 24CF and 25CF solutions). The fulvic acid solutions were dialysed twice against 0. l y HCI, then against distilled water until chloride-free, freeze dried and stored in a desiccator.
Fractionation of humie and fulvie acids (a) Membrane filtration. Samples of 22CH and 22CF were each separated into three fractions of nominal molecular weights 1000-10,000, 10,000-30,000 and greater than 30,000 by membrane filtration as follows. One litre of solutions containing 1 g of either 22CH or 22CF in 0.1M NaCl at p H 7-8 was added to a stirred 1 1. pressure filtration vessel~ fitted with a 3-in. dia. Diaflo PM30 membrane'~ (nominal molecular weight cut-off, 30,000). The cell was pressurized to 6.3 kg/cm 2 through a reservoir containing 1 1. of 0.1M NaC1 which thus replaced solution passing out of the cell. Filtration was continued until 1900 ml of effluent were collected. This effluent was then filtered through a Diaflo P M I 0 membrane (nominal molecular weight cut-off, 10,000), washed through with 500 ml of 0.1M NaCI, and filtration continued until 100 ml of solution remained in the cell. The effluent which had passed through the PM10 membrane was then filtered through a Diaflo UM2 membrane (nominal molecular weight cut-off, 1,000) and washed through with 250 ml 0- 1M NaCl, filtration being continued until 100 ml of solution remained in the cell. The pH of the solutions retained by the PM30, PM10 and UM 2 membranes was adjusted to 2 with 6N HC1. The precipitated humic acids were recovered by centrifugation and the fulvic acid solutions were desalted by washing with water on a Diaflo UM 2 membrane and freeze dried. (b) Gel filtration. Humic and fulvic acids from soil 22 were fractionated by Sephadex gel filtration (small scale) as described by Butler and Ladd (1969a). (c) Gel eleetrophoresis. Preparations 22CH, 22CF and each of their Diaflo fractions were further studied using polyacrylamide gel electrophoresis. Samples (2.5 mg and 7.5 mg for humic and fulvic acids, respectively) were dissolved in 0.5 ml. Tris buffer, pH 8.3 containing 2 0 ~ sucrose. Aliquots (20 ~1) were applied to the top of 30~ gel rods (Davis, 1964) in a Canalco electrophoresis apparatus.+ A current of 4 mA per gel rod (4.0 × 0.5 cm) was applied for 30 rain. The gel rod was extruded and two sections were cut out, one from 1 mm * 22C, 24C, 25C are alkaline extracts from soils 22, 24 and 25 respectively; F and H designate fulvic and humic acids respectively. t Available from the Amicon Corp. Lexington, Massachusetts, U.S.A. ++Available from Canalco, Bethesda, Maryland, U.S.A.
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
251
in front of the salt front to 3 mm behind, and the other, the remainder of the gel rod back to the origin. These sections were then placed in separate centrifuge tubes, the gel crushed with a glass rod, allowed to stand overnight in distilled water and centrifuged. The absorbance at 400 nm and the volumes of the extracts were measured and, after correction for material extracted from a blank section of gel, the percentage distribution of fast moving material was determined.
Properties of humic and fulvic acids Carboxyl contents of the preparation were determined by methylation followed by hydrolysis (Butler and Ladd, 1969a). Ash contents were determined after heating samples in platinum boats for 4 hr at 600°C, carbohydrate contents by the method described by Oades (1967), and amino-acid release by acid hydrolysis and absorbance spectra as previously described (Butler and Ladd, 1969a).
Enzyme substrates and assay procedures The sources of enzymes and substrates* have been described (Ladd and Butler, 1969a, b). Table 1 summarizes protease assay conditions. Fulvic and humic acids were dissolved in 0"025M Tris buffer, pH 7.5 and preincubated with buffered enzyme for either 30 min (carboxypeptidase) or 60 min (other proteases) at the specified temperature. Substrate was TABLE1. ASSAYSOFPROTEASEACTIVITY Enzyme (mg)
Substrate (~.mol)
Pronase (0.1) Trypsin (0.06) Carboxypeptidase (0.005) Papain (0"008)
Z-gly-leu (1.0) BAA (1.0) Z-gly-phe (1.0) BAA (1"0)
Buffer (/~mol) Tris (20) Tris (20) Tris (20) Citrate (40), EDTA (1), mercaptoethanol (10)
Temp. (°C)
pH
Reaction time (rain)
37
7- 5
70
25
7"5
60
25
7.5
30
37
5" 5
60
Total reaction volume: 1 ml added and after the specified reaction time, enzyme activity was stopped with 0.1 ml 5N HCI. Ammonia or amino acid released by enzyme action was determined with a ninhydrin reagent (Moore and Stein, 1954). Values were corrected for those of controls without substrate. Activities in the presence of humic or fulvic acids were expressed as percentages of those of corresponding reaction mixtures without humic compounds. RESULTS
Properties of humic and fulvic acids Figure 1 shows the effect of varying concentrations of three humic acids and three fulvic acids on the activities of four proteases. For each enzyme assay, composite graphs illustrate * Substrate abbreviations: N-benzoyl-L-arginineamide (BAA); N-carbobenzoxy-glycylleucine (Z-gly-leu); N-carbobenzoxy-glycolphenylalanine (Z-gly-phe).
252
J . H . A . BUTLER AND J. N. L A D D
t h e results o f t h r e e s e p a r a t e e x p e r i m e n t s , e a c h o f w h i c h tested t h e effect o f the h u m i c a n d fulvic acids e x t r a c t e d f r o m the s a m e soil. H u m i c a c i d s f r o m different soils b e h a v e d similarly, a n d as a g r o u p , differed f r o m the fulvic acids. H u m i c acids i n h i b i t e d trypsin, p r o n a s e a n d c a r b o x y p e p t i d a s e a n d s t i m u l a t e d 120 IOO
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Fro. 1. The effect of different levels of humic and fulvic acids on the activity of four proteolytie enzymes. Conditions of assay as in Table 1. Enzymes preincubated for 1 hr (0.5 hr with carboxypeptidase) with the humic or fulvic acids before addition of substrate. Reference enzyme activities measured without humic preparations. A 22CH • 22CF [] 24CH • 24CF O 25CH • 25CF TABLE 2. Preparation
PROPERTIES OF HUMIC AND FULVIC ACIDS
Ash (~)
Carbohydrate (%)
Amino acid* (%)
Absorbancet 260 nm 450 nm
22CF 24CF 25CF
10.2 4"2 5-5
2"3 28"2 29-3
2.1 9-3 7.4
23-8 6.4 7.9
22CH 24CH 25CH
1 "0 4"4 7"0
0" 8 4-4 2.7
6.6 12-3 10.6
40.1 27.5 25.6
Carboxyl Uncorr.
m-equiv/g Corr.++
1.60 0.44 0-62
5.5 2-9 2.7
6.4 5.0 4.7
5"95 4-02 3"28
4"4 3.6 3-3
4-8 4.5 4" I
* Percentage amino acid-N released by acid hydrolysis x 6" 25. t Absorbance of solution at 1 mg/ml, pH 7.0. Expressed on an ash-free, carbohydrate-free and amino acid-free basis.
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
253
p a p a i n . F u l v i c acids inhibited trypsin a n d p r o n a s e less t h a n d i d h u m i c acids, h a d little o r no effect on p a p a i n a n d slightly s t i m u l a t e d c a r b o x y p e p t i d a s e . T a b l e 2 shows t h a t several p r o p e r t i e s o f the p r e p a r a t i o n s overlap. C a r b o h y d r a t e a n d acidh y d r o l y s a b l e a m i n o acid contents o f h u m i c a n d fulvic acids f r o m the p o d z o l B h o r i z o n were lower t h a n those o f the c o r r e s p o n d i n g p r e p a r a t i o n s f r o m the t o p soils. F u l v i c acid c o n t a i n e d m o r e c a r b o h y d r a t e a n d less a c i d - h y d r o l y s a b l e a m i n o acid t h a n h u m i c acid f r o m the same soil. F u l v i c acids also were o f higher c a r b o x y l c o n t e n t when expressed on an ash-, c a r b o h y d r a t e - , a n d a m i n o acid-free basis. The effects on enzyme activity o f h u m i c a n d fulvic acids, either within a n d especially between groups, correlates neither with their t o t a l c a r b o x y l c o n t e n t n o r with their contents o f i n o r g a n i c material, c a r b o h y d r a t e a n d acidh y d r o l y s a b l e a m i n o acids.
Fractionation of humic and fulvic acids F r a c t i o n a t i o n o f p o d z o l B h u m i c acid (22CH) a n d fulvic acid (22CF) b y Diaflo ultrafiltration, using three m e m b r a n e s o f different exclusion limits, showed t h a t m o s t o f the TABLE 3. NOMINALMOLECULARWEIGHTDISTRIBUTIONOF A HUMICAND A FULVICACID Preparation
Diaflo ultrafiltration Per cent by weight of nominal molecular weight* 1000-10,000
22CF 22CH
10,000-30,000
78 10
> 30,000
10 4
12 86
Percent by absorbance(260 nrn) 22CF 22CH
75.3 11.3
10.4 5.3
14- 3 83.4
Sephadex gel filtration Per cent by absorbance (260 nm) of nominal molecular weight* < 5000 22CF 22CH
25 40
< 10,000
< 50,000
44 54
55 56
* Nominal values based on manufacturers calibrations. TABLE4.
PER CENT LOW MOLECULAR WEIGHT MATERIAL IN HUMIC PREPARATIONS BY ACRYLAMIDE GEL ELECTROPHORESIS
Preparation 22CF 22CH 22CF 1000-10,000:~ > 30,000 22CH 1000-10,000,* > 30,000
Low molecular weight1" ( ~ )
Recovery (~o)
66 35 72 21 61 24
110 92 93 109 96 98
I" Material travelling with salt front. ~. Fractions obtained by Diaflo ultrafiltration. Percentages calculated from absorbances at 400 nm.
254
J.H.A.
B U T L E R A N D J. N. L A D D
fulvic acid distributed in the nominal molecular weight range 1000-I0,000, whereas most of the humic acid components were of nominal molecular weight greater than 30,000 (Table 3). The results obtained by polyacrylamide gel electrophoresis of humic acid 22CH and fulvic acid 22CF and their Diaflo fractions are shown in Table 4. The fractionation obtained using the small pore polyacrylamide gels (30~ w/v) reflected differences in molecular size rather than differences in charge as there was no separation in the large pore gel (7~ w/v) where the preparations ran with the salt front. Fractionation by Sephadex gel filtration indicated that the humic acid had a higher proportion of low molecular weight components than the fulvic acid (Table 3).
Properties of humic andfuh;ic acidfractions All properties described are of fractions obtained by Diaflo ultrafiltration of humic acid 22CH and fulvic acid 22CF. Humic acid components of increasing nominal molecular weight were increasingly effective as inhibitors of trypsin, pronase and carboxypeptidase activities. Stimulation of papain activity by humic acid 22CH was due solely to its components of relatively high molecular weight (Fig. 2). I00
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FIG.. 2.
The effect of different molecular weight fractions of 2 2 C H and 2 2 C F on the activity of four proteolytic enzymes. Conditions of assay as in Table 1 and Fig. I. O [] A
22CH 1,000-10,000 2 2 C H 10,000-30,000 22CH >30,000
• • •
2 2 C F 1,000-10,000 2 2 C F 10,000-30,000 22CF >30,000
The fulvic acid fraction of highest molecular weight had little or no effect on papain activity. Fulvic acid components of increasing nominal molecular weight were in general increasingly effective inhibitors of trypsin and pronase, although slight stimulation of
IMPORTANCE OF THE MOLECULAR WEIGHT OF HUMIC AND FULVIC ACIDS
255
pronase activities by low concentrations of fulvic acids was obtained. The lowest molecular weight fulvic acid components also stimulated carboxypeptidase activity slightly throughout their concentration range, whereas inhibition was obtained with the other fractions and was related both to their concentration and their molecular weight. The results shown in Fig. 2 also show that in general humic acids of a given molecular weight range have significantly more effect on enzyme activity than fulvic acids of the same molecular weight range. Other properties of the fractions are shown in Table 5. The effects on enzyme activity of the various fractions were not directly correlated with their carboxyl content. TABLE 5. PROPERTIES OF HUMIC AND FULVIC ACID FRACTIONS OBTAINED BY DIAFLO ULTRAFILTRATION
Preparation
Ash (~)
Carbohydrate (%)
Amino acid* (%)
Absorbance'~ 260 nm 450 nm
22CF 1000-10,000 10,000-30,000 > 30,000
23"1 11.1 5.3
1-8 -3-4
2"0 2-0 2"6
22-6 25.2 26"8
22CH 1000-10,000 10,000-30,000 > 30,000
4-2 -4"9
0.2 0.6 0"8
4"6 6"4 7"4
42.8 43-6 36"0
Carboxyl Uncorr.
m-equiv/g Corr.++
1"66 1"95 2"74
3"6 4-1 4"6
4"9 -5.2
6-02 6"80 6"10
4"4 4"1 3"9
4"8 -4"5
* Per cent amino acid-N released by acid hydrolysis × 6.25. t Absorbance of solution of concentration 1 mg/ml, pH 7"0. **Corrected to an ash-, carbohydrate- and amino acid-free basis.
DISCUSSION
The molecular weight distribution of either a humic or a fulvic acid preparation differed according to the technique used. The molecular weights shown (Table 3) are derived entirely from the manufacturers calibrations of Diaflo membranes and Sephadex gels, and in the case of acrylamide gel electrophoresis no calibration was available. Thus molecular weights quoted are nominal and it is not surprising that the values do not agree with many of the widely varying values quoted in the literature (Mehta et al., 1963; Schnitzer and Skinner, 1968; Kononova, 1966). However comparisons between fractions obtained by a given method are valid. Based on absorbance (260 nm) of fractionated material, 54~o of humic acid and 447o of fulvic acid were of molecular weight less than 10,000 when fractionated by Sephadex gel filtration, whereas using Diaflo membrane filtration 119/o of the humic acid and 7 5 ~ of the fulvic acid were of molecular weights 1000-10,000 (Table 3). It is obvious that other factors, e.g. adsorption and exclusion of charged molecules, may be influencing the separations obtained by one or both of the methods. However, the results obtained by membrane filtration are considered to be the more correct. Thus, by this technique fulvic acid 22CF was of lower molecular weight than humic acid 22CH, which is consistent with the results obtained by polyacrylamide gel electrophoresis (Table 4) and which agrees with the general conclusions of other workers (Scheffer and Ulrich, 1960--p. 41; Schnitzer and Desjardins, 1962).
256
J.H.A. BUTLER AND J. N. LADD
The evidence suggests that the Sephadex gel filtration method leads to anomalously high values for the molecular weights of the fulvic acid. Schnitzer and Skinner (1968) also consider that values of molecular weight obtained by fractionation of a podzol B fulvic acid by Sephadex gel filtration were two to ten times higher than those obtained by other methods. Although quantitatively the results of Sephadex gel fractionation of humic acids did not agree well with the Diaflo fractionation figures, acrylamide gel electrophoresis confirmed that humic acid fractions previously obtained with Sephadex (Butler and Ladd, 1969a) graded in molecular weight as expected (Butler, unpublished). Also, the nominated higher molecular weight components of humic acids fractionated by either technique were the more effective stimulators of papain activity and inhibitors of trypsin activity (Ladd and Butler, 1969b--Fig. 2). Despite similar carboxyl contents of all preparations, humic acids have a greater effect on enzyme activity than fulvic acids (Fig. 1) and high molecular weight components of both humic and fulvic acids have a greater effect than the respective low molecular weight components (Fig. 2). In previous papers, Ladd and Butler (1969a, 1970) suggest that changes in enzyme activity with humic acids may result from changes in enzyme conformation due to the enzyme being bound to the humic polymer by a cation exchange mechanism. The extent to which an enzyme molecule is distorted by such a mechanism would be determined in part by the rigidity, size, shape and charge density of the humic molecules. Thus a humic molecule of high molecular weight, because of its inherent rigidity in comparison with an equal weight of smaller molecules should distort the enzyme further. The direct relationship between molecular weight of humic compounds and their effect on enzyme activity is consistent with the effects on proteases of humic and fulvic acids considering their respective molecular weight distributions. However, Fig. 2 shows that humic and fulvic acid fractions of the same molecular weight range differ in their effects on enzymes, and demonstrates that other factors additional to differences in molecular weight distribution are important. Qualitative differences between humic and fulvic acids cannot be stated with certainty because their structure, including the distribution of the carboxyl groups is little known. However, the humic acid polymer is considered to be more aromatic and more condensed than the fulvic acid polymer (Scheffer and Ulrich, 1960--p. 41; Kononova, 1966--p. 91). Hence humic acids should be more rigid than fulvic acids and more able to distort enzyme molecules bound to them. None of the effects on enzymes are related to the ash, amino acid or carbohydrate contents of the humic preparations. This is shown well in the carboxypeptidase assay where all the fulvic acids exert essentially the same effect, yet their ash contents range from 4.2 to 10.2%, their carbohydrate contents range from 2-3 to 29-3% and amino acid-N yields vary from 0- 34 to 1.49%. Acknowledgement--The
authors are indebted to Mr M. AMATOfor technical assistance.
REFERENCES
BUTLERJ. H. A. and LADDJ. N. (1969a) Effect of extractant and molecular size on the optical and chemical properties of soil humic acids. Aust. J. Soil Res. 7, 229-239. BUTLERJ. H. A. and LADDJ. N. (1969b) The effect of methylation of humic acids on their influence on proteolytic enzyme activity. Aust. J. Soil Res. 7, 263-268. DAVISB. J. (1964) Disc electrophoresis--II. Method and application to human serum proteins. Ann. N . Y . Acad. Sci. 121, 404-427. FELBECKG. T. (1965) Structural chemistry of soil humic substances. Adv. Agron. 17, 327-368.
I M P O R T A N C E OF THE M O L E C U L A R WEIGHT OF H U M I C A N D FULVIC ACIDS
257
KONONOVA M. M. (1966) Soil Organic Matter, 2nd English edn, Pergamon Press, Oxford. LADD J. N., BRISBANEP. G. and BUTLERJ. H. A. (1968) The Susceptibility of Nitrogenous Components of Humic Acids to Enzyme Attack: Inhibition of 'Pronase' Activity, Transactions of the Ninth Congress of the International Society for Soil Science, Adelaide, Vol. 3, pp. 319-327. LADD J. N. and BUTLER H. J. A. (1969a) Inhibitory effect of soil humic compounds on the proteolytic enzyme 'Pronase'. Aust. J. Soil Res. 7, 241-251. LADD J. N. and BUTLERJ. H. A. (1969b) Inhibition and stimulation of proteolytic enzyme activities by soil humic acids. Aust. J. Soil Res. 7, 253-261. LADD J. N. and BUTLERJ. H. A. (1970) The effect of inorganic cations on the inhibition and stimulation of protease activity by soil humic acids. Soil Biol. Biochem. 2, 1-8. MEHTA N. C. DUBACH P. and DEUEL H. (1963) Untersuchtmgen uber die Molekularge wichts-Verteilung von Huminstoffen dutch Gelfiltration an Sephadex. Z. PflErndhr. Dang. Bodenk. 102 (2), 128-137. MOORE S. and STEIN W. H. (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. biol. Chem. 211,907-911. NORTHCOTEK. H. (1965) A Factual Key for the Recognition of Australian Soils, CSIRO Australian Division of Soils Divisional Report No. 2/65. OADESJ. M. (1967) Carbohydrates in some Australian soils. Aust. J. Soil Res. 5, 103-116. SCHEFFERF. and ULRICH B. (1960) Humus und Humusdungung, Ferdinand Enke, Stuttgart. SCHNITZER M. and DESJARDINSJ. G. (1962) Molecular and equivalent weights of the organic matter of a podzol. Proc. Soil Sci. Soc. Am. 26, 362-365. SCI-INITZER M. and SKINNER S. I. M. (1968) Gel Filtration of Fulvic Acid, a Soil Humic Compound. In Isotopes and Radiation in Soil Organic-Matter Studies, pp. 41-55, IAEA, Vienna.
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