comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating...
TRANSCRIPT
COMPARISON OF SOME PROPERTIES OF SOIL HUMIC ACIDS AND SYNTHETIC PHENOLIC POLYMERS INCORPORATING AMINO
DERIVATIVES
By J. N. LADD* and J. H. A. BUTLER*
[Manuscript received November 25, 19651
Summary
Twenty-three model phenolic polymers, either nitrogen-free or incorporating amino acids, peptides, or proteins, have been prepared from p-benzoquinone and catechol under mild oxidative conditions. Two lines of experimentation have demonstrated properties of soil humic acids closely similar to those of polymers incorporating proteins, but different from those of polymers incorporating amino acids: (1) fractionation of humic acids and synthetic polymers by "Sephadex" gel filtration showed that the percentage of components of molecular weights nominally greater than 100 000 ranged from 52-76 % for eight humic acids tested, 53-59 % for benzoquinone-protein polymers (excluding polymers containing protamine), but less than 20% for all other polymers; (2) acid hydrolysis with 6~ HCl resulted in a partial release of polymer nitrogen. Amino acid nitrogen in the hydrolysates accounted for 32.4-51.9 % of humic acid nitrogen, 31.2-56.3 % of the nitrogen of polymers incorporating protein, but less than 10.8% of the nitrogen of polymers incorporating individual amino acids.
Experiments with model monomeric N-phenylglycine derivatives and with polymers incorporating simple peptides showed that the bond between the carbon atom of an aromatic ring and the nitrogen atom of an a-amino acid is far more stable to acid hydrolysis than peptide bonds or bonds linking amino acids in humic acids. Glycine is, however, readily released from N-phenylglycine derivatives when conditions favour their oxidation to a quinone-imine intermediate.
Incorporation of proteins into phenolic polymers prevented the detection of peptide bonds by the Folin reagent.
Humic acids, prepared in the usual manner by extraction from soils with dilute alkali or neutral salt solutions, contain varying amounts of nitrogen, of which up to 50% may be accounted for as a-amino acids (Bremner 1955). It has been generally inferred that the amino acids have been derived from proteins either (1) bound as a lignin-protein complex (Waksman and Iyer 1932; Tinsley and Zin 1954; Jenkinson and Tinsley 1960), (2) incorporated during the oxidative polymerization of phenols (Flaig 1955, 1958, 1960, 1964), or (3) combined with quinone groups in the pre-existing humic acid polymer (Burges 1960).
Swaby (1957) and Swaby and Ladd (1962) found no evidence for the presence of peptide bonds themselves in humic acid after a variety of tests, and they proposed that amino acids are incorporated as single units during the oxidative polymerization of phenols. As such they would form an integral part of the humic acid structure,
* Division of Soils, CSIRO, Adelaide.
Aust. J. Soil Res., 1966, 4, 41-54
42 J. N. LADD AND J. H. A. BUTLER
acting, through the cc-amino nitrogen atom, either as bridges between successive aromatic rings or as side chains to the aromatic rings (Hackman and Todd 1953; Mason 1955). Swaby supported his hypothesis by showing that synthetic phenol- amino acid polymers released portions of the incorporated amino acids after hydrolysis with 0.5 or 5~ HC1; some nitrogen remained in the residue.
The validity of Swaby's hypothesis has been further tested using several model phenolic polymers, incorporating amino acids as single compounds, peptides, or proteins. In the present paper, the behaviour of each preparation on acid hydrolysis and the distribution of its components after elution from columns of "Sephadex" gel have been compared with those of humic acids extracted from soil. Since the hypothesis requires amino acids to be bonded in humic acids by an N-phenyl linkage, the stability of this bond has been tested using several monomeric model compounds. Further, the investigations have been extended by an examination of the suitability of the Folin reagent for detection of peptide bonds in humic acids or in synthetic phenolic polymers.
Part of this work has been communicated to the "Technical Meeting on the Use of Isotopes in Soil Organic Matter Studies", held at Braunschweig-Volkenrode, Germany, in September 1963.
11. MATERIALS AND METHODS
(a) Preparation of Soil Humic Acids
Humic acids were extracted from soils with 0 . 5 ~ NaOH in an atmosphere of nitrogen, precipitated by addition of HC1 to pH 1, filtered to remove salts and fulvic acid, redissolved in 0 . 5 ~ Na2C03 or 0 5~ NaOH, and reprecipitated with HC1 a further six times. On three occasions the precipitates were frozen, thawed, and recentrifuged. The humic acids were finally washed three times with 0 . IN HCl and dried in vacuo at room temperature (23°C).
(b) Preparation of Synthetic Polymers Details of the amounts of reactants used, reaction volumes, yields, and nitrogen
contents of the final products are shown in Table 1. The general procedures of preparation were as follows.
(i) Polymers derived from Catechol To solutions of catechol and amino compounds in 0. l~ potassium phosphate,
pH 8.0, was added freshly prepared silver oxide (4 g silver oxide/g catechol). The reaction mixtures were shaken at 25°C for 24 hr, centrifuged, and the dark brown supernatants retained. The precipitates were resuspended in 0 . 5 ~ NaOH (10 ml NaOH/g catechol reactant), stirred for 5 min, recentrifuged, and the supernatants added to those of the first. The alkali-insoluble sediments were discarded. The respective combined supernatants were acidified to pH 1 with 5N HC1, centrifuged, and the supernatants discarded. The black precipitates were frozen, thawed, and recentrifuged. The sediments were again taken up in alkali, centrifuged, the super- natants acidified to pH 1, and the precipitates frozen, thawed, and recentrifuged.
SOIL HUMIC ACIDS AND MODEL PHENOLIC POLYMERS 43
This procedure was repeated a third time, after which the precipitates were washed three times with 0. IN HCl(10 ml HCl/g catechol reactant) and dried at 45°C in vacuo in the presence of caustic soda.
TABLE 1
PREPARATION OF SYNTHETIC PHENOLIC POLYMERS
Weight of Reactants (g) I I
Phenol or Quinone
Catechol 5.0 5.5 5.5 5.5 1 .1 1 .1 2 .2
p-Benzoquinone 5.0 5 .4 5 .4 5.4 5 .4 2.16 2.16 1 .O8 1 .O8 1 .O8 1 .O8 1.08 0.36 1 .O8 2.16 1 .O8 2.16
Reaction
Amino Acid, Peptide, Volume
or Protein 1
- alanine 4.45 glycine casein hydrolysate glycylglycine diglycylglycine protamine sulphate
ammonium chloride alanine glycine casein hydrolysate betaine hydrochloride N-methylglycine DL-alanylglycine glycylglycine glycyl-DL-leucine glycyl-L-tyrosine L-leucylglycine D-leucyl-L-tyrosine digylcylglycine casein ovalbumin protamine sulphate
Polymer Yield*
Polymer Nitrogen
( %) -----
0.00 3.33 4.46 3.51 4.78 3.35 4.13 0.0s' 3 .6Cu 4.25 6.39 4.70 0.29 3.10 4.47 6.90 4.35 3.02 3.68 2.80 6.15 5.55 5.99 3.68
* Percentage of initial reactants excluding oxidants.
(ii) Polymers derived from p-Benzoquinone Solid p-benzoquinone was added to solutions of the amino compounds in
0. l~ potassium phosphate, pH 8.0, and incubated at 45°C for 24 hr. All solutions darkened rapidly on addition ofp-benzoquinone and, within 1-2 hr of commencement of incubation, the pH of the reaction mixtures containing ammonia, amino acids (except betaine), and peptides became acidic. The pH was restored with alkali. The black precipitates formed on subsequent acidification of the reaction mixtures were treated further as described in Section (i).
The nitrogen content of the polymers derived from catechol and amino com- pounds ranged from 3.33 to 4.78 %, and of polymers derived from p-benzoquinone
44 J. N. LADD AND J. H. A. BUTLER
and amino compounds (excluding benzoquinone-betaine polymer) from 2.80 to 6.90 %. Apart from the failure to incorporate the fully-methylated glycine derivative, betaine, there was no obvious relationship between the nitrogen content of a polymer and the nature of the amino compound incorporated, although differences in the molar concentrations of reactants precluded a strict comparison.
Non-nitrogenous polymers were prepared by omitting the amino compounds. Chromatography of concentrated supernatants obtained from cold aqueous
suspensions of the polymer showed that the preparation procedure removed all but faint traces of unreacted amino acids and peptides from the respective polymers. However, the possibility that the benzoquinone-casein and benzoquinone-ovalbumin polymers contained unreacted protein that had coprecipitated with the polymers could not be excluded completely. Two methods of analysis suggested that the proportion of unreacted protein, if any, must be small:
(I) molecular weight distribution of benzoquinone-protein polymers on Sephadex gels (see Section 111), and
(2) electrophoresis on acrylamide gel at pH 9.5. Both polymers moved with the salt front as single, brown, compact bands with faint tails. The amounts of protein in the polymer preparations applied to the gel were at least 4-5 times the minimal amounts for detection with the amido black reagent, yet no protein bands separated from the polymers could be detected.
The benzoquinone-protamine polymer was not tested by gel electrophoresis; but the possibility that the preparation contained unreacted protamine was more remote than that of the other benzoquinone-protein polymers, since protamine is soluble at pH 1 and contains a high proportion of basic amino acids capable of reaction with polymerizing quinones.
(c) Fractionation of Polymers and Humic Acids by Sephadex Gel Filtration The adopted procedure was the same as that described by Posner (1963), except
that sodium chloride was substituted for ammonium chloride as the electrolyte. Columns (15 cm by 1 cm) of Sephadex gels (grades G50 and G100) received 0.4 mg of polymer or humic acid, previously neutralized to pH 7 .O with sodium hydroxide and dissolved in 0.3 m l O . 2 ~ sodium chloride. Distilled water was used as the eluant and recoveries were estimated from the absorbance of fractions at 260 mp, measured on a Shimadzu SV-5OA recording spectrophotometer.
(d) Hydrolysis of Preparations Each preparation (150 mg) was heated with 10 m l 6 ~ HC1 in a sealed tube
containing air at 105°C for 24 hr. The hydrolysis mixtures were centrifuged and the supernatants were retained for estimations of total nitrogen, amino acid nitrogen, and ammonia nitrogen. Residues were washed with 6~ HC1, dried in vacuo at 45"C, weighed, and their nitrogen contents determined.
(e) Analysis of Hydrolysates (1) Total nitrogen was determined by a micro-Kjeldahl method (Bremner 1955).
(2) Amino acid nitrogen was determined colorimetrically, after separating the amino
SOIL HUMIC ACIDS A N D MODEL PHENOLIC POLYMERS 45
acids on paper chromatograms, reacting with ninhydrin, and extracting the coloured product (Block, Durrum, and Zweig 1958). Absorbances measured at 540 mp were related to those of respective amino acid standards or, in the cases of hydrolysates obtained from humic acids or polymers incorporating protein or casein hydrolysate, to the absorbances of standard amounts of casein and ovalbumin hydrolysates. (3) Ammonia nitrogen (including ammonia liberated from amino sugars in the humic acid preparations) was determined by titration after steam distillation of the hydro- lysates made alkaline with sodium hydroxide. (4) Catechol and hydroquinone were measured semiquantitatively by their absorbance at 274 and 290 mp respectively, after elution from paper chromatograms. Diazotized sulphanilic acid was used as a general reagent for the detection of phenols on chromatograms.
(f) Chromatography Solvents
Amino acids were separated or identified using the following solvents (Block, Durrum, and Zweig 1958)
(1) n-butanollacetic acidlwater (4 : 1 : 5, v/v), (2) pyridinelacetic acidlwater (50 : 35 : 15, v/v), (3) pyridinelisoamyl alcohol/water/diethylamine (10 : 10 : 7 : 0.3, vlv), (4) 2,6-lutidine/ethanol/water/diethylamine (55 : 25 : 20 : 2, v/v), (5) phenollwater (400 : 100, v/v).
Phenols were run in solvents, described by Reio (1958), namely, (1) methyl isobutyl ketone14 % formic acid (1000 : 100, v/v), (2) chloroform (containing 1 % ethanol)/methanol/water/formic acid (1000 :
100 : 96 : 4, vlv).
111. RESULTS AND DISCUSSION
(a) Fractionation of Humic Acids and Polymers by Gel Filtration
Eight soil humic acids and fifteen synthetic polymers were fractionated on columns of Sephadex G50 and G100. Figure 1 shows the total percentage recovery and the percentage yield of the higher molecular weight fraction for each preparation. Based on absorbance at 260 mp, recoveries of humic acids from the G50 gel ranged from 96.5 to 105.5% and from the GI00 gel, from 94.4 to 104.7%. Recoveries exceeding 90% were obtained from both grades of gel for polymers prepared from p-benzoquinone with the exception of three preparations, namely, polymers containing either no amino compound or incorporating glycine or alanine alone. Recoveries of polymers prepared from catechol were lower than those of polymers prepared from p-benzoquinone. For either class of polymer, where recoveries were less than 90 %, better yields generally were obtained from the GlOO gel than from the G50 gel. Polymeric material, adsorbed to the gels, remained as a dark brown or black band at the top of the column, even after passage of 10 column volumes of eluant.
Polymers prepared from either p-benzoquinone or catechol and containing amino compounds other than protein were composed of a greater proportion of compounds of relatively low molecular weight than those present in soil This applied to eluted material only since the molecular weights of adsorbed on the gels were unknown.
46 J. N. LADD AND J. H. A. BUTLER
Of the synthetic polymers, only benzoquinone-casein and benzoquinone- ovalbumin showed a molecular weight distribution comparable with those of soil humic acids. This was obvious, not only from measurements based on absorbance at 260 mp, but also from a visual comparison of the relative amounts of brown or black material in each fraction.
Polymer No amino compound benzoquinone
incorporated catechol I benzoquinone-alanine catechol-alanine
Individual amino benzoquinone-glycine acids incorporated catechol-glycine
benzoquinone-casein hydrolysate catechol-casein hydrolysate
r benzoquinone-glycylglycine
catechol-glycylglycine Peptides incorporated
benzoquinone-diglycylglycine
C catechol-diglycylglycine benzoquinone-protamine sulphate
Proteins incorporated benzoquinone-casein benzoquinone-ovalbumin
Soil humic acids
humic acid A humic acid C humic acid J humic acid E humic acid G humic acid K humic acid D humic acid I
Ultraviolet (260 mp) absorbing ma- terial eluted from Sephadex gels
(percentage of input)
Fig. 1.-Fractionation of humic acids and synthetic polymers by Sephadex gel filtration. 0 Material of nominal mol. wt. > 100000, total recoveries from Sephadex GI00 gel, 0 material of nominal mol. wt. > 9000, total recoveries from Sephadex G50 gel.
The increased proportion of black phenolic polymers of relatively high molecular weight obtained with the benzoquinone-protein polymers may be reasonably attri- buted to the incorporation of protein itself into the polymer. To date, fractionations on a scale sufficiently large to permit analyses for total nitrogen and amino acid nitrogen have not been carried out on synthetic polymers. However, soil humic acid J has been fractionated on a Sephadex GlOO column (80 cm by 4.8 cm) and analyses have shown that the high molecular fraction, i.e, components of molecular weight nominally greater than 100000, contained 1.95 times as much nitrogen per gram as that of the
SOIL HUMIC ACIDS AND MODEL PHENOLIC POLYMERS 47
smaller molecular weight fraction, and released 2-66 times as much amino acid per gram on acid hydrolysis. The smaller proportion of phenolic material in the large molecular weight fraction was reflected in a smaller specific absorbance at 260 mp, being only 0.58 times that of the low molecular weight fraction at pH 7.0. The higher specific nitrogen content of the larger molecular weight fraction may be inter- preted as due to either a higher density of reaction sites at which amino compounds have linked on to the phenolic polymer or selective incorporation of protein com- ponents of longer chain length.
(b) Hydrolysis of Soil Humic Acids and Synthetic Polymers A further comparison of the properties of humic acids and synthetic polymers
was obtained from their behaviour on acid hydrolysis (Table 2). All preparations showed several common features but significant differences were obtained in the proportion of amino acid nitrogen released from the polymers.
(i) Amino Acid Nitrogen.-Hydrolysates of synthetic polymers, formed either from catechol or p-benzoquinone, contained progressively increasing proportions of total nitrogen and amino acid nitrogen as the number of peptide bonds in the starting material was increased, e.g. amino acid nitrogen of the hydrolysate accounted for 6.6-10.8 % of the nitrogen of polymers incorporating individual amino acids, for 22 -7-25 a2 % of the nitrogen of polymers incorporating the peptide glycylglycine, for 31 -1-32.9 % of the nitrogen of polymers incorporating the peptide diglycylglycine, and for 31.2-56-3 % of the nitrogen of polymers incorporating proteins. Amino acid nitrogen accounted for 32.4-51.9% of the nitrogen of the natural humic acids tested.
Paper chromatograms of hydrolysates of polymers formed from alanine or glycine reacted with benzoquinone or with catechol and silver oxide showed but one ninhydrin-positive spot, corresponding to the respective amino acid used. By contrast, hydrolysates of polymers formed from alanine or glycine reacted with catechol by the more drastic procedure of Coulson, Davies, and Khan (1959) using persulphate as oxidant contained several ninhydrin-positive compounds, including the respective amino acid used in the polymer preparation. The total ninhydrin reacting material, estimated as a-amino acid nitrogen, accounted for only 5-7 % of the polymer nitrogen.
(ii) Ammonia Nitrogen.-Ammonia was released from all soil humic acids and from all synthetic polymers except those in which the protein, protamine, was incor- porated. Ammonia nitrogen accounted for 4.1-12.3 % of polymer nitrogen, there being no obvious relationship between the amounts of ammonia released and the nature of the amino compounds incorporated into the polymer. Partial deamination of amino acids or peptides during oxidative polymerization of the phenol or quinone (Trautner and Roberts 1951), with subsequent incorporation of the ammonia into the polymer, may explain the presence of ammonia in acid hydrolysates of the polymer. A polymer prepared from p-benzoquinone and ammonia contained 3.60 % nitrogen, 50 % of which was solubilized by acid hydrolysis. However, only 4.1 % of the polymer nitrogen was released as ammonia nitrogen.
(iii) Residue Nitrogen.-The percentage nitrogen content of the residue was less than that of the initial preparation, i.e. hydrolysis released nitrogen as amino acids,
Pol
ymer
or
Hum
ic A
cid
Hum
ic a
cid
D
Hum
ic a
cid
G
Hum
ic a
cid
I H
umic
aci
d J
Cat
echo
l C
atec
hol-
alan
ine
Cat
echo
l-gl
ycin
e C
atec
hol-
case
in h
ydro
lysa
te
Cat
echo
l-gl
ycyl
glyc
ine
Cat
echo
l-di
glyc
ylgl
ycin
e C
atec
hol-
prot
amin
e
Ben
zoqu
inon
e B
enzo
quin
one-
amm
onia
B
enzo
quin
one-
alan
ine
Ben
zoqu
inon
e-gl
ycin
e B
enzo
quin
onee
case
in h
ydro
lysa
te
Ben
zoqu
inon
e-gl
ycyl
glyc
ine
Benzoquinone-diglycylglycine
Ben
zoqu
inon
e-pr
otam
ine
Ben
zoqu
inon
e-ca
sein
B
enzo
quin
oneo
valb
umin
TA
BL
E 2
HY
DRO
LYSI
S O
F H
UM
IC A
CID
S A
ND
S
YN
TH
ET
IC P
HE
NO
LIC
PO
LY
ME
RS
Pol
ymer
N
itro
gen
( %)
Aci
d-so
lubl
e N
itro
gen*
( %
)
Am
ino
Aci
d N
itro
gen*
( %
) -
32
-4
51 -
9
40
.7
35.8
Am
mon
ia
I A
cid-
solu
ble
I R
esid
ue
Nit
roge
n*
Pol
ymer
t N
itro
gen
( %)
* Per
cent
ages
of
tota
l nit
roge
n of
pre
para
tion
. t P
erce
ntag
es o
f w
eigh
ts o
f pr
epar
atio
n in
itia
lly
hydr
olys
ed.
SOIL HUMIC ACIDS AND MODEL PHENOLIC POLYMERS 49
ammonia, and other compounds of low molecular weight without a corresponding destruction of the polymer chain to soluble products. In some cases the percentage of acid-soluble nitrogen was almost four times the percentage of polymer solubilized. Further, calculations based on average molecular weights of 120 and 150 for amino acid and phenolic constituents respectively, showed that humic acids (Table 2) could contain, at a maximum, only six phenolic moieties for each molecule of amino acid actually released by acid hydrolysis. Failure to obtain an extensive breakdown of the humic acid polymers, despite the release of 60.0-66.0% of humic acid nitrogen, suggests that nitrogen was not present exclusively as an integral part of the polymer chain, i.e. as bridging units between phenolic rings, but rather was attached, at least in part, in side chains.
The nature of the hydrolysate nitrogen, other than amino acid nitrogen and ammonia nitrogen, is unknown. Since the hydrolysates vary in colour from light brown to dark brown, part of the hydrolysate nitrogen is probably still linked to phenolic polymers that have been solubilized by the acid treatment.
(iv) Phenol Recovery.-Recoveries of phenols released by hydrolysis of synthetic polymers were very low. Chromatograms of reduced concentrated hydrolysates showed that the yields of catechol and hydroquinone from the respective parent polymer were less than 0 .5 %, irrespective of whether the polymer contained nitrogen. These low recoveries were similar to those obtained by hydrolysis of humic acids (Jakab et al. 1962), but differed markedly from those reported by Coulson, Davies, and Khan (1959), who found that at least one-third of the catechol used in the preparation of a synthetic catechol-glycine polymer was released by acid hydrolysis. Attempts to hydrolyse a catechol-glycine polymer, prepared according to Coulson, Davies, and Khan, again resulted in low recoveries of catechol.
(c) Hydrolysis of Synthetic Polymers incorporating Simple Peptides The incorporation of amino compounds into phenolic polymers involves the
formation of a bond between the amino nitrogen atom and a carbon of the phenolic ring (Mason 1955). The differences observed in the relative amounts of a-amino acids released from the various polymers, described above, indicates a greater resistance to acid hydrolysis of this bond than that of the peptide bond (and of the bonds linking amino acids in humic acids). This was tested by hydrolysing several polymers prepared from p-benzoquinone and simple dipeptides (30 mg polymer/5 ml HC1). Table 3 shows that the relative amounts of amino acids released from the polymers depended upon both the nature of the amino acid and the position of the amino acid in the initial peptide reactant.
For example, glycine nitrogen, released from polymers that had incorporated the peptides, glycylleucine and glycyltyrosine, accounted for only 3.2 and 4.2% respectively of polymer nitrogen, but increased to 20.6 and 25.3% of polymer nitrogen on hydrolysis of preparations that had incorporated the peptides, leucyl- glycine and alanylglycine. Similarly, leucine nitrogen in the acid hydrolysate of polymers that had incorporated the peptides, leucylglycine and leucyltyrosine, accounted for 12.3 and 12.4% respectively of polymer nitrogen, but increased to 20.9 % of polymer nitrogen on hydrolysis of benzoquinone-glycylleucine.
50 J. N. LADD AND J. H. A. BUTLER
Within the range of polymers tested, the results showed that the amino acid released in the greater amount by acid hydrolysis was that acid whose amino group was involved in the peptide linkage in the initial peptide reactant, and therefore was not free to react with the polymerizing benzoquinone. The results agreed essentially with those reported by Flaig (1964).
DISTRIBUTION OF AMINO ACIDS RELEASED BY ACID HYDROLYSIS OF SYNTHETIC POLYMERS INCORPORATING
SIMPLE PEPTIDES
Polymer
1 Polymer Nitrogen in I Amino Acid* 1 Polymer Nitrogen in /
Hydrolysate ( %) i
I (a) ('J) ,
Benzoquinone-alanylglycine Benzoquinone-gl ycylleucine Benzoquinone-glycyltyrosine Benzoquinone-leucylglycine 1
Benzoquinone-leucyltyrosine
* (a) Amino acid of peptide reactant whose a-amino group is not involved in peptidelinkage. (b) Amino acid of peptide reactant whose a-amino group is involved in peptide linkage.
(d) Hydrolysis of N-Phenylglycine Derivatives Studies on the release of glycine from simple monomeric N-phenylglycine
derivatives provided further information for comparison with amino acid release from soil humic acids. Table 4 shows that no glycine was released from N-phenyl- glycine (I) or N-o-carboxyphenylglycine (11) when heated at 105°C for 24 hr with 6~ HC1 in air. Under the same conditions, N-p-hydroxyphenylglycine (111) released 11.8 % of its nitrogen as glycine nitrogen. Whereas solutions of I and I1 remained colourless after hot acid treatment, solutions of I11 were yellow-brown, due presumably to the formation of polymeric substances that were not extractable by benzene.
The results clearly show that the N-phenyl linkage was far more stable to acid hydrolysis than those linkages involving amino acids in soil humic acids; the small release of glycine from compound I11 was probably due to hydrolysis of the quinone- imine derivative, IV, formed during its partial polymerization. Quinoneimines are characteristically unstable towards mineral acids.
In a second series of treatments, hydrogen peroxide was included as an oxidant in the reaction mixtures and caused glycine to be released in increased amounts from
SOIL HUMIC ACIDS AND MODEL PHENOLIC POLYMERS 5 1
all three derivatives, particularly from compounds I and 11. As much glycine was formed at -15OC as at 105OC. However, there was no evidence that polymerization had taken place in any reaction mixture containing hydrogen peroxide, but the evidence was rather that quinones of low molecular weight had been formed. In the case of compound 111, the quinone was purified and found, by elemental analysis and by spectral studies in the ultraviolet, visible, and infrared regions under a variety of conditions, to consist almost entirely of tetrachloro-p-quinone (V). The formation of compound V from compound I11 again indicated the intermediary formation of a quinoneimine before cleavage of the amino acid moiety. None of the treatments caused ammonia to be released from any of the glycine derivatives.
FORMATION OF GLYCINE BY HYDROLYSIS OF IV-PHENYLGLYCINE DERIVATIVES
Compound (10 pmole)
Treatment
Hz02 (0.5 ml 100 vol)
present present absent absent present present absent absent present present absent absent
Temp. ("C)
Glycine Nitrogen ReleasedY
* Expressed as a percentage of total nitrogen of derivative.
(e) Tests for Proteins in Humic Acids and Synthetic Polymers The proposal by Swaby (1957) and Swaby and Ladd (1962) that humic acids
do not contain proteins, arose from the failure to detect peptide bonds by various procedures, including the sensitive method of Lowry et al. (1951), which depends, for a positive response, upon the reduction of a phosphomolybdic-phosphotungstic acid reagent by a copper peptide complex. This reagent is also reduced by phenols (Folin and Ciocalteu 1927), and, as applied to humic acids or phenolic polymers, any response must be corrected for a relatively large absorbance obtained in controls from which copper is omitted.
Eight humic acids, containing 1.91-3 -62 % nitrogen, were tested by this method. The observed response per unit nitrogen content of the sample varied with each preparation. Four preparations gave weak positive tests for protein, three gave negative results, i.e. a negative value for the change in absorbance, and one showed
J. N. LADD AND J. H. A. BUTLER
Fig. 2.-Examination of humic acids and synthetic polymers for the presence of peptide bonds. 1, Diglycylglycine; 2, ovalbumin; 3, casein; 4, humic acid C ; 5, humic acid J ; 6 , humic acid E; 7, humic acid A; 8, benzoquinone-casein polymer; 9, benzoquinone-ovalbumin polymer; 10, humic acid G; 11, benzo-
quinone-diglycylglycine polymer.
no change. Figure 2 shows the results obtained with five of the humic acids. Further, this figure shows clearly that the incorporation of a peptide or protein into a phenolic polymer markedly interfered with its ability to react with the protein reagent. Hence it may reasonably be concluded that the absence of a positive protein response using this method does not negate the presence of peptide bonds in the humic acid preparation.
SOIL HUMIC ACIDS AND MODEL PHENOLIC POLYMERS 5 3
IV. CONCLUSIONS It is not suggested that the synthetic model compounds studied here accurately
represent soil humic acid in all respects. Two simple compounds, catechol and p-benzoquinone, have been chosen as the basic units, both capable of polymerization and incorporation of amino compounds. The extent to which such a process contri- butes to the incorporation of amino compounds into soil humic acids is unknown. Assuming, however, that basically similar reaction mechanisms operate in the incorporation of amino compounds into humic acids and the model compounds, it must be concluded that the amino acid components of humic acids are not derived exclusively from reactions of quinones with amino acids themselves.
Results show that synthetic phenolic polymers, in which peptides and proteins are incorporated, represent humic acids more accurately than those incorporating amino acids. This is based in part on the proportion of polymer nitrogen released as a-amino acid nitrogen on acid hydrolysis (Table 2). Evidence obtained with polymers incorporating simple peptides (Table 3) and model monomeric glycine derivatives (Table 4) indicates that amino acids, bonded by their a-amino group to aromatic rings, are much more stable to acid hydrolysis than amino acids linked in humic acids. Further, only those polymers that have incorporated proteins (excluding the protein, protamine, of relatively low molecular weight) show a distribution of components on columns of Sephadex gel comparable with that of humic acids.
However, the formation of polymers incorporating amino acids or proteins need not be mutually exclusive for humic acid formation (Flaig 1960). If humic acids are formed by the polymerization of various phenols and quinones, it seems reasonable that opportunities will exist for the incorporation of amino acids, peptides, and proteins. Formation of polymers of the first type may partly account for the nitrogen of the acid-resistant residues of humic acids.
Unequivocal evidence for peptide bonds in humic acids has still to be obtained. Scharpenseel and Krausse (1962) have reported the liberation of 14C-labelled amino acids from %-labelled humic acids after incubation with the protease, papain, but the method of obtaining the labelled humic acids was not described. Application of the Folin reagent to humic acids showed only a weak positive response by a few of the preparations tested (Fig. 2). However, the failure of the benzoquinone-protein polymers to give a positive response with the Folin reagent or to react with the staining reagent, amido black, shows that the peptide bonds are masked when incorporated into phenolic polymers. The susceptibility of peptide bonds to biological attack is currently being studied using polymers in which proteins are incorporated either during or after phenol polymerization.
V. ACKNOWLEDGMENTS The authors are indebted to Dr. R. J. Swaby for many helpful discussions and
for gifts of humic acids, to Dr. R. W. L. Kimber for his assistance in identifying tetrachloro-p-quinone, to Mr. B. Cartwright for running samples of benzoquinone- protein polymers on acrylamide gels, and to Mr. M. Amato for general technical assistance.
J. N. LADD AND J. H. A. BUTLER
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