Encapsulation of protein in humic acid from a histosol
as an explanation for the occurrence of organic nitrogen
in soil and sediment
Xu Zang, Jasper D.H. van Heemst, Karl J. Dria, Patrick G. Hatcher *
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USAAbstract
Recent work suggests that nitrogen in humic acids exists primarily as amide functional groups that mirror those in protein. However, the mode for the existence of such labile materials as protein still remains unclear. With the com-bined applications of NMR spectroscopy, tetramethylammonium hydroxide (TMAH) thermochemolysis, pyrolysis/ GC/MS, and elemental analysis, we propose that the survival of proteins in humic acids is carried out by encapsulation into hydrophobic domains of humic acids. To test this hypothesis, we simulated encapsulation of15N-labeled protein
extracts into humic acids and demonstrated that complete hydrolysis of the protein is prevented by the encapsulating humic acid. Results from this study constitute evidence to support the encapsulation mechanism involved in the for-mation of refractory organic nitrogen during sediment diagenesis.#2000 Published by Elsevier Science Ltd. All rights reserved.
Keywords:Refractory organic nitrogen; Protein survivability; Encapsulation; Humic acid; TMAH GC/MS; Pyrolysis GC/MS; Solid-state NMR
1. Introduction
The profound in¯uence of humic materials on the distribution, bioavailability, and ultimate fate of sedi-mentary organic nitrogen in various environmental sediments has been recognized in the past decade (Bar-ancikova et al., 1997; Schulten and Schnitzer, 1997; Lichtfouse et al., 1998; Stankiewicz and van Bergen, 1998). Sedimentary organic nitrogen (SON) refers to the sum-total of all nitrogen-containing substances in sedi-ments. SON provides most of the nitrogen necessary for the terrestrial bioproductivity. It primarily results from the microbial decay of proteins and peptides in bioor-ganic residues and is part of the nitrogen cycle in the biosphere. During early diagenesis, most of the labile nitrogen-containing materials, such as peptide, protein, and chitin, are quickly degraded and mineralized by microbial and/or enzymatic degradation (Codispoti and Christensen, 1985; Blackburn and Sorensen, 1988; Endo et al., 1995; Nguyen and Harvey, 1998; Walton, 1998).
However, recent studies have shown that part of these labile nitrogen-containing materials are incorporated into refractory organic components in humic sub-stances, where they are protected from biodegradation (Hedges and Keil, 1995; Henrichs, 1995; Knicker and Hatcher, 1997; Nguyen and Harvey, 1998; Knicker et al., 1999). This incorporated organic nitrogen has been removed from the active nitrogen pool of the nitrogen cycle and is, therefore, no longer available for biological production (Knicker and Hatcher, 1997). Estimates of refractory organic nitrogen in humic substances range from 30 to 50% of total nitrogen (Schulten and Schnit-zer, 1997). It is essential to understand the mechanisms and processes involved in the stabilization of labile organic nitrogen in humic substances to improve our knowledge of the chemical composition of the refractory organic nitrogen and understanding of the nitrogen cycling in the biosphere.
A number of analytical techniques have been applied to reveal the identity of the refractory organic nitrogen in humic substances. Due to the chemically complex and physically heterogeneous system, it has been dicult to
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obtain accurate structural information on the organic nitrogen in humic substances. The advent of modern analytical methodologies, namely solid state nuclear magnetic resonance (NMR) spectroscopy, pyrolytic methods coupled to gas chromatography and mass spectrometry (Py/GC/MS), and the tetramethylammonium hydroxide (TMAH) thermochemolytic methods (TMAH/ GC/MS), have provided a wealth of new structural information on the dierent types of organic moieties in humic substances (Knicker et al., 1996; Schulten and Schnitzer, 1997; del Rio et al., 1998). Recent applica-tions of solid-state 15N NMR to the study of dierent
types of organic matter in soil systems at natural 15N
abundance levels have shown that the main function-ality of the refractory organic nitrogen in humic acid exists primarily as amide functional groups, probably from proteins (Knicker et al., 1996; Knicker and Hatcher, 1997; Derenne et al., 1998). The amide functional group persistently exists even after intense chemical and microbial degradation. Pyrolytic methods coupled to analytical techniques, such as gas chromatography and mass spectrometry, have oered additional structural information and have identi®ed proteinaceous pyrolysis products from humic acid (Bracewell and Robertson, 1984; Zsolnay and Harvey, 1985; Gadel and Bruchet, 1987; Schulten and Schnitzer, 1997). Furthermore, the application of TMAH thermochemolysis for the struc-tural analysis of the peptide-like refractory organic nitrogen in humic substances has resulted in a more comprehensive evaluation of structural entities of the amide-nitrogen and con®rmed that it is derived from proteinaceous material (Knicker and Hatcher, 1997). Heterocyclic nitrogen-containing compounds, such as pyrrole and indole, are also part of the refractory organic nitrogen, but they only constitute minor pro-portions (less than 5% of the total refractory organic nitrogen) (Schulten and Schnitzer, 1997).
Although various studies have revealed that the nitrogen-bearing compounds in humic acid are most likely proteins, less well understood and far more con-troversial are the roles played by various mechanisms in the association of humic acid and refractory organic nitrogen. The depolymerization-recondensation hypothesis was proposed to explain the existence of the refractory organic nitrogen (Tissot and Welte, 1984; Bada, 1998). This mechanism speculates that the formation of organic nitrogen matter involves an initial degradation of cellular components and a subsequent recondensation of these degradation species with other low molecular weight compounds, resulting in new substances, which cannot easily be related to their biological precursors. In this process, naturally occurring macromolecules such as proteins and carbohydrates are enzymatically degra-ded to oligomers and monomers, which for the most part are mineralized. A small fraction of these oligomers and monomers may condense or polymerize into
organic rich colloids by chemical (Maillard, 1912; Flaig et al., 1975; Hedges, 1978; Keil and Kirchman, 1994) or photochemically initiated crosslinking reactions (Harvey et al., 1983). A well known example is the condensation reaction between amino acids and carbohydrates to form Schi bases, which subsequently undergo Amadori rearrangements to form dark colored melanoidins (Maillard, 1912). However, solid-state15N NMR studies
do not con®rm the presence of melanoidins as the major nitrogen-containing compounds in humic substances (Knicker et al., 1996) excluding this mechanism as the primary preservation mechanism. In contrast to the depolymerization-recondensation mechanism, the selective preservation mechanism claims that refractory organic matter is a fraction of cell components which have not been degraded due to the inherent resistance of the compound itself to enzymatic or chemical attack, whereas the other more labile constituents are mineralized in the water column and the upper layer of sediments (Hatcher et al., 1983; Moers et al., 1994; Derenne and Largeau, 1998; Lichtfouse et al., 1998). Therefore, macromolecular structures such as aquatic kerogen and algaenan have been selectively preserved during early diagenesis and have survived microbial decomposition. Nevertheless, proteins, the largest compartment of nitrogen in most cells, and thus the greatest potential contributor of nitrogen to environmental sediments, are rapidly lost during early diagenesis and, therefore, are an unlikely substrate for a selective preservation mechanism.
Adsorption of intrinsically labile organic matter to mineral surfaces, which are inaccessible to bacterial extracellular enzymes (<10 nm), has been proposed as another mechanism for the formation of refractory organic matter. According to this mechanism, labile materials are adsorbed to mineral surfaces and stabi-lized by incorporation into mineral pores (mesopores) that are too small to allow entry and/or eective enzyme function (Mayer, 1994, 1995; Salmon et al., 1998). While mineral adsorption may be one controlling factor on organic nitrogen distributions, there are other processes that are also important for the preservation of organic matter (Henrichs, 1995; Mayer, 1995; Knicker et al., 1996; Knicker and Hatcher, 1997). Proteinaceous mate-rials have been identi®ed in clay-free compost and humic substances with minor amounts of mineral con-stituents, and mineral or clay adsorption appears to be an unlikely event in these cases.
1993) and in soil microaggregates (Skjemstad et al., 1993). Organic-rich deposits, generally low in mineral constituents, have also shown excellent long-term pre-servation of protein (Knicker et al., 1996). Based on previous studies, the basic macromolecular framework of humic substances can be described as highly branched networks of aliphatic structures with varying degrees of aromaticity (Hatcher et al., 1980; Hatcher et al., 1981; Almendros et al., 1996). The aliphatic components of the network in humic substances may play an important role in encapsulating proteins or their high molecular weight counterparts and protecting them from biological and chemical degradation over geological time scales.
1.1. Objective of this study
In this study, the nature of the refractory organic nitrogen in humic acid from an organic-rich sediment (Everglades peat) was investigated for any proteinaceous materials encapsulated within the humic acid structure. Furthermore, 15N-labeled proteins were added to the
humic acid to simulate an encapsulation process in order to elucidate the mechanism that may be respon-sible for the preservation of labile organic nitrogen in humic acid. The fate of the labeled organic nitrogen during the chemical hydrolysis of the humic acid was followed by solid state NMR spectroscopy, pyrolysis/ GC/MS, and TMAH thermochemolysis GC/MS.
2. Materials and methods
2.1. Everglades peat humic acid
Everglades peat humic acid was prepared as pre-viously described (Hatcher et al., 1986). A core was obtained from sawgrass peat at a depth of 65±70 cm at a site adjacent to Alligator Alley, west of Hialeah, Florida. The core sample was air-dried to constant weight and subsequently extracted with a 2:1 benzene:ethanol mix-ture in a Soxhlet apparatus to remove bitumens. The bitumen-extracted peat was treated with 0.1 N HCl at room temperature to remove carbonates and other low molecular weight acid-soluble components. After washing and removing the acid by centrifugation and decanta-tion, the wet peat was treated with 0.5 N NaOH under nitrogen to extract the humic acid. After centrifugation and decantation of this extract, extraction was repeated with 0.5 N NaOH, and this extract was collected and combined with the ®rst extract. The alkaline extracts were passed through glass ®ber ®lters to remove sus-pended materials, and acidi®ed with concentrated HCl to a pH=2. The humic acids were allowed to precipitate overnight. Subsequently, centrifugation isolated the fulvic acids from the humic acids, which were washed several times with 0.1 N HCl and lyophilized.
2.2. 15N labeled algae
A mixed algal culture was prepared as previously described (Zelibor et al., 1988). Strains of Chlorella, Chlamydomonas,Closterium andScenedesmus, isolated from algal mats overlying peat deposits in Conservation District 2, The Everglades (Florida), were mixed in equal proportions and grown in a liquid medium con-taining 15N-enriched potassium nitrate. Algal cells in
mass culture were harvested during exponential growth using a Sorvall RC2-B centrifuge equipped with a KSB continuous ¯ow system (Ivan Sorvall, Inc., Norwalk, CT). Cells were washed several times with sterile dis-tilled water and then lyophilized. A more detailed description of the algal cells growth is given by Zelibor (Zelibor et al., 1988).
2.3. Simulated encapsulation procedure
Approximately 40 ml of 0.5 M NaOH was mixed with
15N (100%) labeled dry algae for 20 min to extract algal
proteins. Ten milliliters of the extraction solution were mixed with 300 mg of Everglades peat humic acid in a Falcon tube at room temperature and left for 2 weeks with constant shaking. After 2 weeks of shaking, the humic acid±algal protein solution was acidi®ed to pH 1.5 using 12 M HCl. After centrifugation, the pre-cipitated residues were frozen at ÿ5C for 1 day and
subsequently lyophilized. The dried residues were sub-sequently hydrolyzed using 6 M HCl under re¯ux for 6 h. The hydrolyzed residues were thoroughly washed with water 3 times, followed by lyophilization. These dried residues were referred to as HHAP residues.
2.4. Flash pyrolysis/gas chromatography/mass spectrometry
Pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS) analyses were performed using a Carlo Erba mega 500 series gas chromatograph operating in split mode, equipped with a Chemical Data System (CDS AS-2500) pyrolysis interface and a 30 m fused silica capillary column coated with chemically bound DB 50 (0.25 mm i.d., ®lm thickness 0.10mm).
Approxi-mately 0.5 mg samples were weighed and transferred to quartz tubes (CDS Analytical Inc.). The samples were heated at a rate of 5C/ms to an indicated temperature
of 610C after being placed onto the heated (280C)
pyrolysis interface for 15 s under a high ¯ow of He to purge the pyrolysis chamber. Helium was used as a carrier gas with a ¯ow rate of 0.88 ml/min to sweep the volatiles into the fused silica capillary column. The following oven temperature program was used: initial temperature 40C (1 min), increased by 20C/min to 110C, 7C/min
to 240C, 15C/min to 310C, held at 310C for 10 min.
MS25 RFA mass spectrometer ®tted with a Kratos Mach 3-Dart data system. Pyrolysis products were identi®ed based on their mass spectra and GC retention time.
2.5. TMAH thermochemolysis/gas chromatography/ mass spectrometry
Samples (2±3 mg) were weighed and placed in glass ampoules with 200mL of TMAH (25% in methanol).
After 30 min mixing, the methanol was evaporated under N2gas. The ampoules were sealed under vacuum
and heated at 250C in an oven for 30 min. After cooling,
the tubes were cracked open and a known volume of ethyl acetate (100ml) that contained an internal standard
(39 ng/mL of n-eicosane) was added to the tubes. All
inside surfaces were washed (3 times) using ethyl acetate. The combined extracts were reduced to approximately 200ml under a stream of N2. The diluted samples (1ml)
were analyzed by capillary gas chromatography±mass spectrometry (GC±MS) on a Hewlett-Packard 6890 gas chromatograph operating in split mode, equipped with a 15 m fused silica capillary column coated with chemically bound DB-5 (0.25 mm i.d., ®lm thickness 0.1 mm,
Supelco, Bellefonte, PA). Helium was used as a carrier gas with a ¯ow rate of about 1 ml/min. Temperature conditions were initial temperature at 40C for 1 min to
320C at 15C /min then held at 320C for 10 min. The GC
was directly coupled to a Pegasus II (Leco Corporation, St. Joseph, MI) time-of-¯ight mass spectrometer by a deactivated fused silica transfer-line heated to 300C.
Mass spectra from 33 to 400m/zwere accumulated at a scan rate of 2 kHz, summed and recorded at 9 scans/s. Most peaks were identi®ed by comparison with the NIST (version 1.6) library.
2.6. Solid-state15N NMR
Solid-state 15N NMR spectrometry utilizing cross
polarization magic angle spinning (CP-MAS) and TPPM (two pulse phase modulation) was performed using a Bruker DMX-400 MHz nuclear magnetic resonance (NMR) spectrometer with a1H frequency of 400 MHz or a15N
resonance frequency of 40.5 MHz. The spectrum of 100 mg
15N labeled algae, 100 mg Everglades peat humic acid,
100 mg hydrolyzed Everglades peat humic acid (HHA), and 98 mg hydrolyzed Everglades peat humic acid-protein (HHAP) were obtained from 128, 259000, 114000, 114000 accumulated scans, respectively, with a pulse delay of 250 ms, a contact time of 1 ms, and a magic angle spinning speed of 5 kHz. The chemical shifts were plotted using glycine as a reference (ÿ375.80 ppm).
2.7. Solid-state13C NMR
Solid-state13C NMR spectrometry of Everglades peat
humic acid utilizing cross polarization magic angle
spinning (CP-MAS), a ramp-CP procedure, and TPPM (two pulse phase modulation) was performed using a Bruker DMX-300 MHz nuclear magnetic resonance (NMR) spectrometer with a1H frequency of 300 MHz or
a13C resonance frequency of 75.48 MHz. The spectrum
of 80 mg humic acid was obtained from 32000 accumu-lated scans, with a pulse delay of 1 s, a contact time of 1 ms, and a magic angle spinning speed of 13 kHz. The chemical shifts were plotted using the carboxyl signal of glycine as external standard (176.03 ppm).
2.8. Elemental analysis
The CHN contents of humic acid and treated humic acid residues were determined by standard combustion analysis at the microanalysis laboratory of Atlantic MicroLab. Inc. (Norcross, GA, USA).
3. Results and discussion
3.1. Everglades peat humic acid
3.1.1. Solid-state15N and13C NMR of humic acid
The 13C NMR spectrum of Everglades peat humic
acid (Fig. 1) is characterized by major structural com-ponents observed in many other peats (Orem and Hatcher, 1987). The signal characteristic of paranic carbon at 33 ppm stands out as an important signal. Methoxy (lignin) and alkoxy (polysaccharides) carbons yield signals at 56 ppm and 72 ppm, respectively. Signals between 100 and 129 and at 172 ppm are characteristic of aromatic and carboxyl carbons, respectively. Aliphatic and aromatic carbons represent 64 and 22%, respec-tively, of the total organic carbon in Everglades peat humic acid. Fig. 2 shows the solid-state 15N NMR
spectra of the Everglades peat humic acid indicating a
Fig. 1. Solid-state 13C NMR spectrum of Everglades peat
predominantly proteinaceous nature (Jacob et al., 1980; Witanowski et al., 1993; Knicker and LuÈdemann, 1995; Knicker et al., 1996). A small signal at ÿ80 ppm may
indicate pyridinic nitrogen. The dominant signal observed atÿ257 ppm is assigned mostly to amide-type
nitrogen. Acetylated amino sugars, lactams, indoles and carbazoles may also contribute to the intensity of this signal (Witanowski et al., 1993). Another signal that also indicates the presence of proteinaceous materials is the signal atÿ343 ppm, which corresponds to nitrogen
of terminal amino groups of peptides (Witanowski et al., 1993). The solid-state 15N NMR spectrum of the
hydrolyzed Everglades peat humic acid residue is also shown in Fig. 2. Although a signi®cant decrease in the signal was observed, probably due to the loss of proteins associated with the humic acid, the signals corresponding to amide and amino groups are still identi®able in the spectrum. These ®ndings are in contrast with the con-sensus that the formation of refractory organic nitrogen in sediments is due to a condensation reaction (Maillard reaction) between carbohydrates and amino acids during the humi®cation process (Maillard, 1912; Benzing-Purdie et al., 1986; Qian et al., 1992; Yamamoto and Ishiwatari, 1992). Compounds from such condensation reactions have been shown to yield pyrrolic N (ÿ145 to
ÿ240 ppm), and pyridinic N (ÿ40 to ÿ90 ppm) as
major resonance signals in the solid-state 15N NMR
spectrum of a model study (Benzing-Purdie and Rat-clie, 1986). However, solid-state 15N NMR spectra of
humic acid residue presented here show a totally dif-ferent signal pattern. No major, but perhaps minor, signals in the region of typical melanoidinic-nitrogen from the Maillard condensation reactions were observed. Similar results from other sedimentary humic substance systems were also obtained (Knicker and Hatcher, 1997), where dominant amide and amino sig-nals were shown as the major nitrogen resonance lines (Knicker et al., 1999).
3.1.2. Py/GC/MS of humic acid
Hydrolyzed humic acid (HHA) residues, those expected to contain refractory organic nitrogen, were pyrolyzed at 610C and analyzed by gas chromatography/mass
spectrometry. Mass chromatograms and the corre-sponding mass spectra of the ionsm/z154 and 168 are shown in Fig. 3. The group of ions withm/z154 and 168 are often used as distinctive markers for proteinaceous materials pyrolysis products and have been proposed as being diketopiperazine compounds derived from dipep-tides structures (Munson and Fetterolf, 1987; Smith et al., 1988; Stankiewicz et al., 1998). These proteinaceous pyrolysis products (diketopiperazine derivatives) have been identi®ed in pyrolysis products of model dipep-tides, human hair, bone proteins, and gelatin (Ratcli et al., 1974; Langhammer et al., 1986; Munson and Fet-terolf, 1987; Smith et al., 1988; Chiavari and Galletti, 1992; Stankiewicz et al., 1996; Stankiewicz et al., 1998; Poinar and Stankiewicz, 1999). In Fig. 3, a group of compounds displaying the prominent ions of m/z 154 are derived from the dipeptides Pro-Gly, Pro-Lys, Pro-Val and Pro-Arg. The group of compounds displaying the prominent ions ofm/z168 are likely derived from the di-peptide Pro-Ala (Stankiewicz et al., 1996). Due to the low natural abundance of 15N (0.37%), a search for the
diketopiperazine derivatives using characteristic ions for
15N-containing products (m/z156, 170) failed to reveal
the presence of these products in the pyrograms of HHA residue. These pyrolysis results provide com-plementary evidence to NMR results which indicate the presence of proteinaceous materials in the humic acid hydrolysis residue. This may mean that the amide reso-nance shown in the 15N NMR spectrum of Fig. 2 is
mostly derived from proteinaceous materials.
It is also important to point out the presence of ali-phatic components in the pyrolyzate of a hydrolyzed humic acid residue (Fig. 4). This pyrolyzate revealed the presence of a prominent homologous series of aliphatic
molecules (alkanes and alkenes) as was observed pre-viously for whole peat (Kotra and Hatcher, 1988). It has been suggested that these aliphatic components are concentrated in humic acid by a process of selective preservation during early diagenesis (Hatcher and Orem, 1986).
3.1.3. TMAH of humic acid
Fig. 5 shows a reconstructed mass chromatogram of the tetramethylammonium hydroxide (TMAH) thermo-chemolysis products obtained from the hydrolyzed Everglades peat humic acid. The TMAH thermo-chemolysis reaction yields methylated derivatives of speci®c amino acids from proteins. The identi®ed amino
acid methyl esters with the m/z of their base ion and possible origins are indicated in Table 1. The mass chromatogram was constructed by usingm/z58+59+ 72+73+100+101+84+85+116+117+98+99+116+ 117+103, which corresponds to them/zof each14N and 15N-containing amino acid methyl ester base peak
(shown in Table 1). TMAH appears to disrupts the 3-dimensional structure of humic acid, to release entrapped proteinaceous materials and subsequently hydrolyzes and methylates them. Identi®cation of the products was facilitated by the TMAH analysis of a series of di-, tri-, and tetrapeptides of known structure (data not shown). In Fig. 5, the partial GC/MS chromatogram reveals various amino acid methyl esters that have proteinaceous origin. Most of the amino acids identi®ed in this study represent aliphatic amino acids. Although it is possible that other types of amino acids might also be incorporated into humic acid, our results here might imply a preferential preservation of aliphatic amino acids. A complete study on the reaction and yield of TMAH and each individual amino acid is currently carried out to further investigate this preliminary hypothesis.
3.2. Nature of refractory organic nitrogen in Everglades peat humic acid
The solid-state15N NMR spectrum of the humic acid
suggests a proteinaceous nature for this refractory organic nitrogen in humic acid. The presence of amide-nitrogen in humic acid was persistent, even after intensive acid hydrolysis. Py/GC/MS and TMAH/GC/MS analyses con®rm that this refractory organic nitrogen is mostly protein or its high molecular weight counterparts such as peptide or structurally modi®ed proteins. It is likely that proteinaceous materials have survived early diag-enesis and have been protected within the structure of humic acids. We speculate that the preservation of the labile proteinaceous materials is due to a physical pro-tection within the three-dimensional structure of the humic acid. The paranic carbon components identi®ed in13C NMR, Py/GC/MS and TMAH/GC/MS analysis
might play important roles in the preservation of pro-teinaceous materials. Wershaw et al. (1986) presented a model for humic acid in which they suggest that the structure of humic acid resembles micelle-like aggre-gates. It is similar to biological lipoprotein membranes in that they are comprised of hydrophobic interiors and hydrophilic exterior surfaces. Labile compounds can be imbedded in the micelle-like structure and be protected by the hydrophobic interiors and hydrophilic exteriors. Therefore, proteinaceous moieties might be physically encapsulated within the aliphatic moieties of humic acid. The hydrophobic environment of the aliphatic moieties protects the encapsulated proteinaceous materials from chemical and/or microbial degradation.
Fig. 3. (A) Mass chromatograms of the characteristic ionsm/z
3.3. 15N Labeled algal materials
In order to test the encapsulation hypothesis, 15N
labeled algal proteins were mixed with humic acid in alkaline solution and then co-precipitated to simulate the encapsulation process. The formation of15N refractory
organic nitrogen by this encapsulation mechanism was monitored by15N NMR, Py/GC/MS and TMAH/GC/
MS. We used algal proteins as labeled nitrogen markers to indicate the fate of organic nitrogen during chemical decomposition of the humic acid-protein mixture. If humic acid indeed encapsulates the added15N labeled
Fig. 4. Mass chromatogram of the characteristic ions of alkanes and alkenes (m/z55+57+69+71+83+85) present in the pyrolyzate of the hydrolyzed humic acid (HHA).
Fig. 5. Mass chromatogram of the TMAH thermochemolysis products released from the hydrolyzed humic acid (HHA). The mass chromatogram was constructed withm/z58+59+72+73+100+101+84+85+116+117+98+99+116+117+103, which corre-sponds to them/zof each15N and14N amino acid methyl ester base ions in the mass spectra (Table 1). Compounds were identi®ed by
proteins, solid-state 15N NMR, Py/GC/MS and
TMAH/GC/MS should reveal the15N enrichment in the
refractory organic nitrogen in humic acid. Various fac-tors, such as incubation time of humic acid with 15N
proteins, size of the proteins, and the hydrophobicity of the protein fragments, may play critical roles in the encapsulation process and the eects of these processes will be evaluated in future studies. We incubated the extracted proteins with humic acid under alkaline con-ditions for two weeks for several reasons. First, the long incubation time increased the association between the proteins and the humic acid. Second, the 2-week incu-bation under alkaline conditions can partially hydrolyze the protein into smaller fragments. The hydrolyzed fragments that have smaller size might be preferred to be encapsulated into the humic acid structure.
3.3.1. Solid-state15N NMR of algae
The solid-state 15N NMR spectrum of 15N labeled
algae is shown in Fig. 6. Similar spectra were obtained from the algae Chlorella (Knicker et al., 1996). The predominant amide signal atÿ257 ppm corresponds to
peptide-nitrogen due to the fact that proteinaceous materials constitute the bulk of algal biomass. The resonance signals at ÿ294 and ÿ304 ppm are mostly
assigned to the amino groups of basic amino acids or nucleosides (Witanowski et al., 1993). The resonance signal corresponding to the free amino group atÿ345
ppm was also resolved.
3.3.2. Py/GC/MS of15N labeled proteins
Diketopiperazine derivatives from algal proteins were identi®ed by the Py/GC/MS analysis of the15N labeled
algal proteins. The reconstructed mass chromatograms and the corresponding mass spectra for these pyrolysis products of15N labeled algal proteins are shown in Fig.
7. The elution pattern observed for the diketopiperazine
derivatives from algal proteins was very similar to that of the diketopiperazine derivatives from the HHA residue. Because the algal proteins were15N-labeled, the masses
of all of the dipeptide diketopiperazine derivatives that contain two15N atoms are twom/zunits higher than the
masses of the corresponding 14N-containing
diketopi-perazine derivatives produced upon pyrolysis of the HHA residue. Mass chromatograms ofm/z154 and 168 failed to identify any 14N-containing diketopiperazine
derivatives, indicating that most of the nitrogen incor-porated in proteins is15N labeled.
Table 1
Amino acid methyl esters identi®ed in the thermochemolyzate of the hydrolyzed humic acid residue with them/zof the base ion of each amino acid ester and their possible origins
m/z
Compound 14N 15N Possible origin
Glycine-N-dimethyl, methyl ester 58 59 Glycine
Alanine-N-dimethyl, methyl ester 72 73 Alanine
Valine-N-dimethyl, methyl ester 100 101 Valine
Proline-N-dimethyl, methyl ester 84 85 Proline
Leucine(isoleucine)N-dimethyl, methyl ester 116 117 Leucine/isoleucine
5-Hexenoic acid,2-methylamino,methyl ester 98 99 Lysine
Proline- 1-methyl-5-oxo, methyl ester 98 99 Aspartic acid
3-Phenyl-2-propenoic acid, methyl ester 103 N/A Phenylalanine
Phenylalanine, N-dimethyl, methyl ester 116 117 Phenylalanine
Fig. 6. Solid-state 15N NMR spectrum of freeze-dried 15N
3.3.3. TMAH/GC/MS of15N labeled proteins
A mass chromatogram revealing the presence of TMAH thermochemolysis products released from algae is shown in Fig. 8. The mass chromatogram was constructed by the same method used previously for the TMAH mass chromatogram of the HHA residue. The identi®ed amino acid methyl esters were detected in thermochemolysis products from the 15N labeled
algal protein. Because the proteins were 15N labeled,
the mass fragments utilized for all of the amino acid esters are 1 m/z unit higher than the mass fragments of the corresponding amino acid ester from the HHA residue.
3.4. Simulated encapsulation experiments and hydrolyzed humic acid-15N protein residues (HHAP)
3.4.1. Procedures of the simulated encapsulation The15N labeled algal proteins were added to humic acid
under alkaline conditions to simulate an encapsulation process. It is known that humic acid displays dierent three-dimensional macrostructures under varying pH values (Flaig and Beutelspacher, 1954; Chen and Schnitzer, 1976; Chen et al., 1978; Ghosh and Schnitzer, 1980; Tan, 1985; Senesi et al., 1992; Chien and William, 1998; Jones and Tiller, 1999). An increasing pH will solublize humic acid, ionize it, and expand it into a
Fig. 7. (A) Mass chromatograms of characteristic ions of diketopiperazine derivatives present in the pyrolyzate of15N labeled algal
proteins. The 15N nitrogen is indicated by an asterisk. (B) Mass spectra of diketopiperazine derivatives with m/z 156 and 170
chainlike primary structure. Under these conditions, the hydrophobic and/or ionic interactions between humic acid and charged proteins are favored and protein molecules become associated with humic acid, but encapsulation of proteins into the expanded humic acid structure is unlikely. When the pH is decreased to pH 1±2, humic acid becomes protonated and its hydrophobicity increases, leading to a three-dimensional structure by aggregation and cross-linking. During this aggregation, the associated proteinaceous molecules can become incorporated into the three-dimensional structure, where they are physically entrapped within the hydrophobic humic acid structure. These proteinaceous molecules are no longer chemically available to acid and they are protected from chemical hydrolysis.
3.4.2. Solid-state15N NMR of humic acid-protein
residue (before and after acid hydrolysis)
The solid-state 15N NMR spectrum of humic
acid-mixed with algal protein before and after HCl hydro-lysis (HAP and HHAP) is shown in Fig. 9. Proteins may be strongly adsorbed onto the highly hydrophobic humic acid surface. Consequently, the NMR spectrum of the HAP residue shows a dominant amide signal at
ÿ256 ppm and a small free amino signal atÿ345 ppm,
which correspond to the proteinaceous materials from the
15N labeled algae. The signal-to-noise for this spectrum is
greatly enhanced over natural abundance spectra due to the 15N enrichment. The humic acid-protein (HAP)
sample was subjected to 6 M HCl hydrolysis and washed extensively with water to remove surface-bound proteins. The solid-state 15N NMR spectrum of this
hydrolyzed humic acid-protein residue (HHAP) shown in Fig. 9 revealed a persistent amide signal at-256 ppm and a signi®cant increase of the free amino signal at
ÿ345 ppm. When compared to the solid-state15N NMR
spectrum of the hydrolyzed humic acid residue (HHA, Fig. 2), a signi®cant increase of amide and amino signals is observed in the HHAP NMR spectrum. This clearly indicates the enrichment of the 15N labeled refractory
organic nitrogen in the humic acid structure. The 15N
amide-nitrogen might be incorporated into the three-dimensional hydrophobic matrix network in humic acid by this simulated encapsulation process and become refractory to acid hydrolysis due to the protection from the humic acid hydrophobic network structure. Fig. 8. Mass chromatogram of the TMAH thermochemolysis products released from the15N-labeled algal proteins. The mass
chro-matogram was constructed withm/z58+59+72+73+100+101+84+85+116+117+98+99+116+ 117+103, which corresponds to them/zof each15N and14N amino acid methyl ester base ions in the mass spectra (Table 1). Compounds were identi®ed by
com-parison with the mass spectra of TMAH thermochemolysis products released from model peptides (data not shown).
Fig. 9. Solid-state 15N NMR spectrum of Everglades peat
humic acid mixed with15N-labeled algal proteins before and
It is interesting to note the conspicuous increase in the free amino signal after acid hydrolysis. We interpret this to indicate that, if the proteinaceous materials were indeed encapsulated into the humic acid structure, it is likely that only portions of the long peptide chain are entrapped into humic acid, while other parts of the pep-tide chain may still protrude outside of the humic acid
structure. Therefore, when the HAP residue undergoes 6 M HCl hydrolysis, the encapsulated protein fragments are protected by the humic acid three-dimensional hydro-phobic structure, being inaccessible to HCl and leaving the amide bond uncleaved. The protein fragments outside of the three-dimensional structure are still chemically avail-able and are, therefore, hydrolyzed by the 6 M HCl. As a
Fig. 10. (A) Mass chromatograms of characteristic ions of diketopiperazine derivatives present in the pyrolyzate of the HHAP resi-due: the upper two braces (a)m/z170 and 168 are characteristic ions for15N-labeled and non-labeled pro-ala, respectively; the lower
two braces (b)m/z156 and 154 are characteristic ions for15N-labeled and non-labeled pro-gly, pro-lys, pro-val or pro-arg,
respec-tively. The15N nitrogen is indicated by an asterisk. (B) Representative mass spectra of diketopiperazine derivatives in pyrolyzates of
result, the NMR spectra show the persistent amide signal with decreased peak height and the free amino signal with increased peak height.
3.4.3. Py/GC/MS of hydrolyzed humic acid-proteins residue
Mass chromatograms and the corresponding mass spectra for the HHAP residue are presented in Fig. 10. The diketopiperazine derivative ions (m/z154 and 168) originating from the unlabeled proteins in the humic acid residue were clearly detected in the pyrolyzates. In addition, it is interesting to note the occurrence of a similar group of peaks with m/z 156 and 170 having identical retention times as those mentioned above (Fig. 10). Apparently, both 14N-diketopiperazine derivatives
and15N-diketopiperazine derivatives are present in the
HHAP residue, and compared with pyrolysis products of the unamended HHA residue, there is a signi®cant increase in the amount of15N labeled diketopiperazine
derivatives. This observation is supported by the mass spectra of the diketopiperazine derivatives encountered in the pyrolyzates of the HHAP residue (Fig. 10). The
14N diketopiperazine derivatives (originating from the
HHA residue) produce the characteristic ions ofm/z154 and 168 (Fig. 3), and the 15N diketopiperazine
deriva-tives (originating from the 15N labeled algal proteins)
produce the characteristic ions ofm/z156 and 170 (Fig. 7). In the mass spectra of the diketopiperazine deriva-tives from the pyrolyzate of the HHAP residue (Fig. 10), in addition to the 154 and 168 ion fragments, we observe signi®cant ion fragments at m/z 156 and 170. These represent the ion fragments from 15N-labeled
diketopiperazine derivatives and these ions fragments (m/z 156 and 170) are derived from the 15N-labeled
proteins. Therefore, both of the mass chromatograms
and mass spectra have demonstrated the enrichment of the
15N-labeled proteins in the HHAP residue. This
enrich-ment is most likely a result of the encapsulation process.
3.4.4. TMAH thermochemolysis/GC/MS of hydrolyzed humic acid-protein residue
The TMAH/GC/MS mass chromatogram of the HHAP residue is shown in Fig. 11. The mass chromato-gram was constructed in a similar manner as the pre-vious method used for the mass chromatograms of the thermochemolyzates of the HHA residue (Fig. 5) and
15N algal proteins (Fig. 8). Brie¯y, the mass
chromato-gram was obtained by summing the base peaks of each
15N and14N amino acid methyl ester. In Fig. 11, peaks
appear for all of the previously identi®ed amino acid methyl esters in the thermochemolyzates of the HHAP residue. The reconstructed partial mass chromatograms of the proline and lysine methyl esters released from the HHAP residue is shown in Fig. 12 with their mass spectra shown in Fig. 13. The intensity of each ion trace was plotted on the same scale and represents the abso-lute digital counts obtained for each respective ion. In Fig. 12C and D, the natural abundance (14N) amino
acid methyl esters (m/z 84 and 98) released from the HHA predominate compared to the 15N amino acid
methyl esters (m/z85 and m/z99) are shown. In Figs. 12A and B, the15N-enriched derivatives become more
signi®cant as shown by an increase in the peaks atm/z 85 and 99. This trend can also be seen in the mass spectra (Fig. 13) of the amino acid methyl esters in the thermo-chemolyzate of the HHAP residue, where the ion frag-ments atm/z85 and 99 of HHAP residue were signi®cantly increased, compared to the unamended humic acid.
In addition to proline and lysine derivatives, the enrichment of15N in each of the individual amino acid
derivatives was observed (Fig. 14). In this ®gure, each column represents the ratio of the peak area of 15N
amino acid methyl ester to the peak area of14N amino
acid methyl ester in the mass chromatograms of HHA and HHAP residues. The shaded bars represent the ratio in the thermochemolyzate of the HHAP residue and the open bars represent the ratio in the thermochemolyzate of the HHA residue. The increasing ratio in HHAP over HHA residues indicates that we have successfully encapsulated the labeled proteins and protected them as portions of their structure from hydrolysis.
It is worthy to note that there is one exception: no signi®cant increase in the nitrogen isotope ratio is observed for phenylanaline. This suggests that paranic carbon chains are likely to be involved in the encapsula-tion process. In other words, the hydrophobic interiors of the micelle-like structure of humic acid are probably made of paranic carbon chains. We speculate that, during the incubation process, the protein fragments that have a majority of aliphatic amino acid components (Ala, Gly, Val, etc.) have stronger interactions with the Fig. 11. Mass chromatogram of the TMAH thermochemolysis
products released from the hydrolyzed humic acid-mixed with
15N-labeled algal proteins. The mass chromatogram was
con-structed withm/z58+59+72+73+100+101+84+85+116+ 117+98+99+116+117+103, which corresponds to the m/z
of each15N and14N amino acid methyl ester base ions in the
paranic carbon chain of humic acid than the frag-ments that have a higher content of aromatic amino acids (Phe, Tyr, Trp, etc.). The aliphatic protein fragments are conformationally preferred by the hydrophobic interiors of the micelle-like structures (paranic carbon chain), while protein fragments, which have a higher content of aromatic components, are less associated with the paranic carbon portions of humic acid. Therefore, during the humic acid three-dimensional structure aggregation process, the paranic hydro-carbon portions of humic acid encapsulate the already closely associated aliphatic protein fragments and sub-sequently protect them from chemical and/or microbial degradation, while the less strongly associated aromatic protein fragments protrude outside of the paranic carbon chain network and are cleaved by the subsequent acid hydrolysis. However, more data is still necessary to con®rm this speculation.
3.5. Elemental analysis
The elemental CHN contents of the dierent humic acid residue are listed in Table 2. The amount of nitrogen increased when humic acid was mixed with protein due to the association of protein with humic acid, either adsorbed to the surface of humic acid or encapsulated into the humic acid structure. After 6 M HCl hydrolysis, although half of the nitrogen was lost, nitrogen was still present, accounting for approximately 2% of the organic matter. In the HHAP residue a greater amount of refractory nitrogen (2.46%) was observed compared to the HHA residue (2.13%). The increase of N in the HHAP residue simply arises from the simulated encap-sulation process as we added N-rich protein extracts. The greater nitrogen content in the HHAP residue suggests that externally added algal protein was encapsulated into the humic acid structure.
Fig. 12. Mass chromatograms of lysine and proline methyl esters released from the hydrolyzed humic acid-mixed with15N-labeled algal
proteins (HHAP) and the hydrolyzed humic acid (HHA).m/z98 and 99 are characteristic ions for non-labeled and15N-labeled lysine-methyl
3.6. Discussion of the encapsulation mechanism
As discussed in the beginning of this article, various mechanisms have been proposed as the pathway for the preservation of organic matter in soils and sediments. The present study indicates that proteins may be pre-served in dierent environments by physical encapsula-tion within refractory organic matter, humic acids being an example. Proteins can be directly protected by entrapment into the humic acid matrix (Fig. 15). By changing the pH of the humic acid solution, the con-formational structure of humic acid was modi®ed to associate and entrap externally added algal proteins that carried15N tag. At high pH values, humic acid mainly
possesses a chain-like primary or extended structure. Various protein molecules can become associated with the humic acid by hydrophobic and/or ionic interac-tions. When the pH of the humic acid-protein solution is decreased, the humic acid structure becomes aggregated and forms a 3-dimensional structure. Those associated proteinaceous molecules that are more strongly sorbed
to humic acid maybe entrapped within the 3-dimen-sional structure and are protected against chemical and/ or enzymatic attack. If the proteins are partially entrapped, the entrapped portions are resistant to che-mical hydrolysis; however, the portions external to the humic acid structure are still susceptible to chemical hydrolysis and are degraded by the 6 M HCl hydrolysis. Aliphatic moieties in humic acid might play an important role in the survival of the encapsulated pro-teins during chemical hydrolysis. Previous studies have demonstrated the presence of signi®cant amounts of aliphatic moieties in many humic substances from dif-fering environmental settings (Hatcher et al., 1980, 1981; Orem and Hatcher, 1987; Almendros et al., 1996; Barancikova et al., 1997; Cook and Long®rd, 1998; Lichtfouse et al., 1998; Zhang et al., 1999). The Py/GC/ MS and 13C NMR analyses conducted in the present
study also reveal various aliphatic moieties containing long carbon chains in Everglades peat humic acid. Fig. 13. Mass spectra of proline and lysine methyl ester
deri-vatives released upon TMAH thermochemolysis of the hydro-lyzed humic acid-mixed with 15N-labeled algal proteins
(HHAP), the hydrolyzed humic acid (HHA), and the 15
N-labeled algal proteins.
Fig. 14. Enrichment of15N in various amino acid methyl esters
released upon TMAH thermochemolysis of the HHAP residue. The shaded bars represent the ratio of the amounts of the15
N-labeled amino acid methyl esters to the amounts of non-N-labeled
14N amino acid methyl esters, calculated from the
correspond-ing peak areas. The open bars represent the correspondcorrespond-ing ratio in the thermochemolyzate of the HHA residue.
Table 2
Elemental compositions of humic acid and its treated residues
C% N% H%
Humic acid (Everglades peat) 49.95 4.05 5.45
Humic acid-mixed with algal proteins 52.85 4.12 5.73
Hydrolyze humic acid (HHA) 57.99 2.13 5.27
Hydrolyzed humic acid-mixed with algal proteins (HHAP)
When proteins are physically entrapped into this refrac-tory organic matter (aliphatic moieties), the hydro-phobic aliphatic matrix network can protect the proteins from chemical or microbial attack, resulting in the preservation and survival of labile proteins in humic substances.
4. Conclusions
1. Proteinaceous materials represent an important contributor to refractory organic nitrogen present in humic acids.
2. By changing the conformational structure of humic acid, we have successfully recreated new refractory organic nitrogen in humic acid, and this newly created refractory organic nitrogen is of a proteinaceous nature, derived from externally added15N-labeled algal protein.
The newly formed proteinaceous materials in humic acid are physically encapsulated within the humic acid structure, probably by the aliphatic moieties in humic acid, and are resistant to chemical hydrolysis. These ®ndings constitute evidence to support the encapsu-lation mechanism involved in the formation of refrac-tory organic nitrogen during sediment diagenesis. While adsorption to minerals or clays may be one important factor that accounts for the preservation of organic nitrogen in sediments, proteinaceous moieties or high molecular weight counterparts can be physically
encapsulated into the refractory organic matter in soil and sediments and consequently preserved from micro-bial and chemical degradation.
Acknowledgements
We thank Dr. Heike Knicker (Technische UniversitaÈt MuÈnchen) for valuable discussions and Dr. Joseph Sachleben for assistance in obtaining the solid-state NMR spectra. Financial support for this work was provided by the National Science Foundation (OCE-9711596 and DEB-9727057).
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