Geochimica et Cosmochimica Acta, Vol. 65, No. 9, pp. 1467–1480, 2001
Copyright © 2001 Elsevier Science Ltd
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Pergamon
PII S0016-7037(00)00621-9
Preservation of protein in marine systems: Hydrophobic and other noncovalent associations
as major stabilizing forces
RENO T. NGUYEN† and H. RODGER HARVEY*
Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, MD 20688, USA
(Received February 1, 2000; accepted in revised form November 27, 2000)
Abstract—The fate of proteins during early diagenesis was investigated in environments with low mineral
content to assess preservation mechanisms other than mineral sorption. Preservation was examined in anoxic,
organic-rich sediments of Mangrove Lake, a marine environment located in Bermuda, and for particulate
material generated during oxic decay of diatoms. N-phenacylthiazolium bromide (PTB) treatment tested the
hypothesis that proteins may undergo modification reactions with glucose to form advanced-glycation end
products (AGEs). A small but significant release (additional 14%) of proteins was observed after PTB
treatment in surficial sediments, indicating that some aggregations can proceed through an ␣-dicarbonyl
intermediate of the AGE pathway. Size-exclusion high-pressure liquid chromatography with protein fluorescence, absorbance, and evaporative light-scattering detector measurements under native (phosphate or bicarbonate buffers) and denaturing (guanidine 䡠 HCl, urea, or acetonitrile) conditions point to the importance of
hydrophobic and other noncovalent interactions in the stabilization of proteinaceous material in the environment. Soluble aggregates of substantial, relative molecular mass (Mr ⲏ 106) appear to be formed early in the
diagenetic sequence. The preferential preservation of very high Mr, multisubunit phytoplankton proteins in
sediments suggests that such aggregations confer resistance to degradation. Alternatively, some of the
proteinaceous material may represent that fraction of organic matter that is highly prone to aggregations.
Extended incubations (18 h; 37°C) with trypsin and proteinase-K showed that much of the aggregates that
could be extracted are receptive to proteolytic cleavage. Buffer-, surfactant-, and NaOH-extractable aggregates
comprised most of the acid-hydrolyzable proteinaceous material in detritus and surficial sediments but ⬍35%
in 9.7-m-deep sediments, suggesting additional mechanisms for preservation might be in operation. The
results are direct evidence for the preservation of peptide linkages in sediments as old as 4000 yr and for
noncovalent associations (hydrophobic interaction, hydrogen bonding) of protein as important mechanisms in
long-term preservation. Copyright © 2001 Elsevier Science Ltd
tions. Currently the mechanisms that retain nitrogen-containing
macromolecules, particularly proteins, in the geosphere remain
controversial (Bada, 1998; Stankiewicz and van Bergen, 1998).
Although evidence for protein condensation products in the
environment is scarce, the Maillard reaction is well documented in the biochemical literature (Njoroge and Monnier,
1989). The pathway of this reaction is believed to involve
proteins that undergo modification reactions with glucose and
other reducing sugars to form a class of products termed
advanced-glycation end products (AGEs) or Amadori products.
These Amadori products subsequently may undergo dehydration by successive ␤-eliminations to form an ␣-dicarbonyl
intermediate, which is subject to nucleophilic attack by specific
amino acid groups (amine of lysine or histidine; sulfhydryl of
cysteine) of another protein to form a stable protein–protein
cross-link. The compound N-phenacylthiazolium bromide
(PTB) was used to break one type of such glucose-derived
protein–protein cross-link considered an important marker of
several diseases (Vasan et al., 1996). The application of PTB to
environmental samples was recently realized, with nucleic acids released from within a macromolecular network containing
AGE cross-links in fossilized fecal matter (Poinar et al., 1998).
The importance of noncovalent interactions to protein stability in biologic systems is well known. In cells, proteins are
normally found in their native (i.e., folded) conformation as
monomers or as multimers containing several polypeptides,
which are both stabilized by electrostatic, hydrogen-bond, and
1. INTRODUCTION
The abundance of proteins in living organisms (Voet and
Voet, 1990; Bada, 1998; Lourenço et al., 1998) has sparked
considerable interest into their fates in the aquatic environment.
Historically, proteins have been considered very labile and
consequently unlikely to survive as high molecular mass components during early diagenesis. Evidence is increasing, however, that some fraction of proteinaceous material is preserved
in freshwater, estuarine, and marine environments (Tanoue,
1995; Tanoue et al., 1996; Nguyen and Harvey, 1997, 1998;
Fogel and Tuross, 1999; Pantoja and Lee, 1999). The substantial fraction (up to 60%) of residual nitrogen in algal detritus
and in sediments that can be attributed to high molecular mass
(⬎2 kDa) proteinaceous material indicates the important contribution of proteins to the geosphere (Nguyen and Harvey,
1997, 1998). Although the concept of sorptive preservation
(Keil et al., 1994; Mayer, 1994; Hedges and Keil, 1995) can
help to explain much of the distribution of organic carbon in
oceanic sediments, recent work (Nguyen and Harvey, 1998)
has suggested that other mechanisms also may contribute to
preservation, including the Maillard reaction (Schiff-base condensation) or other cross-linkings and hydrophobic interac*Author to whom correspondence should be addressed (harvey@cbl.
umces.edu).
†
Present address: Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA.
1467
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R. T. Nguyen and H. R. Harvey
hydrophobic interactions (Voet and Voet, 1990; Creighton,
1993; Yanchunas et al., 1994; Jiang et al., 1997). Hydrophobic
associations of integral membrane proteins with membrane
lipids are important also (Voet and Voet, 1990) and may protect
these proteins from proteolytic attack (Tanoue et al., 1995;
Laursen et al., 1996; Suzuki et al., 1997; Borch and Kirchman,
1998; Nagata et al., 1998). Whether in cells or in solution,
partially unfolded (i.e., folding intermediates) or improperly
folded proteins are known to aggregate as exposed hydrophobic
regions randomly associate (Copeland, 1994; Bollag et al.,
1996; Jiang et al., 1997; Herbst et al., 1998). Sulfhydryl mispairing of cysteine residues under oxidizing conditions may
contribute to aggregation (Copeland, 1994), but this appears to
be a secondary factor (Bollag et al., 1996). For solubilization or
denaturation (i.e., unfolding), many multisubunit proteins, integral membrane proteins, and protein aggregates require detergents, organic solvents, or chaotropic agents that disrupt
hydrophobic interactions and hydrogen bonding to different
extents (Voet and Voet, 1990; Creighton, 1993; Nave et al.,
1993; Copeland, 1994; Bollag et al., 1996).
Here we describe the application of PTB and denaturation
agents to evaluate two mechanisms for protein preservation,
glucose-derived protein–protein cross-linking and noncovalent
interactions. Samples ranging from intact algal cells, a degraded detritus of diatomaceous origin, and a sediment section
of preserved algal sapropel were subjected to the various treatments. Size-exclusion chromatography (SEC) coupled to an
evaporative light-scattering detector (ELSD) was applied for
the first time (to our knowledge) for the analysis of proteins.
Our objectives were to follow the formation, preservation, or
both of aggregates during early diagenesis and to elucidate
possible mechanisms for their existence. Incubations with proteases were performed to assess whether the extracted material
remaining after extensive degradation could be receptive to
enzymatic attack, a key measure of protein integrity.
2. MATERIALS AND METHODS
2.1. Application of a Glucose-Derived Protein–Protein CrossLink Breaker
PTB was synthesized according to the method of Vasan et al. (1996).
Identity was confirmed by particle-beam liquid chromatography–mass
spectrometry (HP 59980B-5989A LC/MS) in both electron impact
(major ions: 120, 105, 85, 77, 58, 57, and 51 m/z) and positive chemical
ionization (major ions: 204 [minus bromide ion], 121, 105, 86, and
59 m/z) modes. Purity was assessed by melting-point determination
and by reverse-phase high-pressure liquid chromatography (HPLC)
with a gradient solvent system comprised initially of 10% methanol
in water, increasing linearly to 100% methanol over a period of 35
min at a flow rate of 1 mL/min. Sample elution is monitored by
absorbance at 215 nm and with an Alltech Varex MKIII ELSD (3.35
L/min nebulizer gas [N2] flow rate; 116°C drift tube temperature)
connected in series.
Assessment of AGE cross-links was made on surficial and 9.7-mdeep (⬃4000 yr old) sediments of Mangrove Lake, Bermuda. Detailed
descriptions of the lake and sediments have been reported (Hatcher,
1978; Hatcher et al., 1982; Boudreau et al., 1992; Nguyen and Harvey,
1998). Briefly, Mangrove Lake is a small coastal lake presently with
brackish to saline waters. Sediments are anoxic and characterized by a
low mineral content (⬍10% w/w), owing to the high input of marine
algae and low terrigenous input. For controls and treatment samples,
triplicates of sediment (6 mg lyophilized material per screw-cap microcentrifuge tube) were subjected to extraction with 10% (w/v) trichloroacetic acid in acetone (containing 0.1% ␤-mercaptoethanol) as
described by Nguyen and Harvey (1998). Extraction was continued
with acetone (containing 0.1% 95-mercaptoethanol; three times), followed by drying of the pellet containing cell debris and protein. One
milliliter of 0.2-␮m filter-sterilized PTB incubation buffer (20 mM
PTB, 40.5 mM Na2HPO4, 9.5 mM NaH2PO4, pH 7.4) or the sodium
phosphate buffer alone was added to each treatment sample or control,
respectively, followed by vortex mixing for 1 min and incubation of the
tubes at 37°C for 24 h on a rotary shaker. PTB was prepared fresh in
buffer for each application to avoid oxidation. Duplicates of six to
seven different concentrations (20 –500 ␮g/mL) of protein standard
(ribulose-1,5-bisphosphate carboxylase [RuBisCo]) were also incubated in parallel in either the treatment or control buffers.
After the 24-h incubation period, 300 ␮L of 0.5 N NaOH was added
to each sample and standard. Initial experiments found that PTB and its
oxidation products could interfere with standard protein quantitation
methods; thus, proteins were precipitated with trichloroacetic acid
(10% w/v final concentration; 4°C, 1 h), followed by washing of the
protein pellets with ⫺20°C methanol three times to remove residual
PTB and any oxidation products. We used 1.3 mL of 0.1 N NaOH to
solubilize the protein in each sample and standard tube. After centrifugation (16,000 ⫻ g, 5 min), an aliquot was analyzed by the bicinchoninic acid (BCA) assay and by o-phthaldialdehyde–HPLC (OPAHPLC) of amino acid hydrolyzates (Cowie and Hedges, 1992; Nguyen
and Harvey, 1994). The remaining material was frozen at ⫺70°C.
To assess the potential effect of the sediment matrix on PTB treatment, calibration curves also were constructed for the incubation of
PTB and control buffers with RuBisCo-spiked sediments. Treatment
sets used microcentrifuge tubes, each containing 2 mg of lyophilized,
9.7-m-deep Mangrove Lake sediments and spiked with RuBisCo prepared in pH 7.5 deionized water. Control sets were prepared similarly
but amended with only the water. The standards were vortex mixed
with the sediment, frozen at ⫺70°C, lyophilized, and then subjected to
trichloroacetic acid and acetone extraction. Incubations with and without PTB and subsequent steps for further purification of protein were
performed as described above. Sufficient protein (100, 200, and 300 ␮g
RuBisCo) was added to bracket the concentration range of protein
(0.05– 0.15 g/g dry sediment) found in Mangrove Lake sediments, and
protein was quantified by the BCA assay. Protein extraction efficiency
was determined on the basis of concentration of protein in buffer
relative to that in whole sediments, i.e., [buffer-soluble protein]/[solidphase protein]. The solid-phase protein was determined by amino acid
analysis of lyophilized material after 6 N HCl hydrolysis (150°C, 70
min) (Nguyen and Harvey, 1998).
Shifts in the apparent relative molecular mass (Mr) of proteinaceous
material after PTB treatment was followed with SEC at room temperature with two detectors connected in series. The first was a Waters 474
fluorescence detector with excitation at 280 nm and emission wavelengths set for tryptophan (340 nm) or tyrosine (305 nm). The second
was a Waters 486 UV-Vis absorbance detector set at 280 nm. Monitoring at 280 nm is typical for protein detection because tryptophan and
tyrosine each have a strong extinction coefficient at that wavelength
(Copeland, 1994). A Bio-Prep SE 1000/17 column (8 ⫻ 300 mm;
15–20 ␮m particle size) and an eluent of sodium phosphate buffer (81
mM Na2HPO4, 19 mM NaH2PO4, 150 mM NaCl, 10 mM NaN3, pH
7.4) pumped at a flow rate of 0.5 mL/min was used for the separations.
An additional precolumn filter (0.5 ␮m) provided column protection.
Because the NaOH used for the final protein solubilization step
would ionize tyrosine and shift its absorption maximum (Copeland,
1994), samples for SEC analysis were treated to be at approximately
neutral pH.
Thus, samples were thawed, vortex mixed, and centrifuged, and the
supernatant was treated with trichloroacetic acid (20% w/v final concentration) to induce protein precipitation; the pellet was then washed
twice with ⫺20°C ethanol before solubilization in sodium phosphate
buffer pH 7.4. Samples were centrifuged (16,000 ⫻ g, 10 min), the
supernatants transferred to 250-␮L autosampler vial inserts, and injections (150 –200 ␮L) from two to three replicates made with a Waters
717plus autosampler. A control blank and a treatment blank were
processed in parallel. Commercially available spinach RuBisCo and
SEC standards were used for calibration.
Protein in marine systems
2.2. Testing Effects of Sample Processing on SEC Protein
Elution
Various extraction schemes and elution conditions were evaluated
for differences in protein separations on the SEC column. In addition,
proteins were extracted from fresh or lyophilized (⫺70°C stored)
Thalassiosira weissflogii cells at logarithmic or late-stationary phase of
growth. The following extraction protocols were applied: 0.1 N NaOH
extraction (1 h at 25°C); and the trichloroacetic acid/acetone extractions followed by solubilization in buffer. Buffers included 50 mM
NH4HCO3 (pH 7.8) or 8 mol/L urea prepared in 50 mM NH4HCO3.
SEC separations were performed with the same solubilization solution
or buffer as eluent, with protein elution monitored by fluorescence.
2.3. Evaluation of ELSD for Protein Analysis
To determine if ELSD could be used for quantitative detection of
proteins separated by SEC, all protein calibrants and vitamin B12 (at
10 –180 ␮g per standard) were tested by preparation in 50 mM
NH4HCO3, with the same volatile buffer used as eluent (0.5 mL/min).
ELSD nebulizer gas flow rate and drift tube temperature were 3.5
L/min and 120°C, respectively.
2.4. Determination of Noncovalent Aggregations
In addition to sediment samples, late-stationary-phase Thalassiosira
weissflogii cells and detritus derived from Skeletonema costatum (from
a mesocosm experiment under oxic conditions; Mannino and Harvey,
in preparation) were analyzed. For these samples, extraction size was
increased to provide protein for four different treatments and for
multiple SEC injections. Briefly, 0.5 g of lyophilized material per
sample was weighed into a 50-mL Teflon centrifuge tube, and the
trichloroacetic acid and acetone extractions (with 0.1% ␤-mercaptoethanol) were performed with 40-mL solvent volumes and by 2 ⫻ 5
min ultrasonication (30 W) efforts on ice (after Nguyen and Harvey,
1998). The extracted material was dried under vacuum, and protein was
solubilized in 15 mL of sodium phosphate buffer with NaN3. Tubes
were incubated on a rotary shaker for 48 h at 37°C, then stored at 4°C
until analysis.
To assess noncovalent interactions, proteins were separated by SEC
on the basis of molecular mass (or more accurately, hydrodynamic
volume) under “native” conditions and subsequently under three different denaturing conditions. For each treatment, 1 mL of the phosphate-buffer-solubilized protein, corresponding to ⬃500 ␮g to 5 mg
total protein, was concentrated by trichloroacetic acid precipitation
before solubilization in 200 ␮L of native or denaturing buffers. Solubilization of protein could be achieved by grinding the pellet with a
pipette tip, by vortex mixing, and by brief ultrasonication (30 W, 30 s).
(A) Native conditions involved solubilization of protein in 50 mM
NH4HCO3, with the same buffer used as eluent. (B) Acetonitriledenaturing conditions involved initial solubilization of protein in 50
mM NH4HCO3, followed by addition of pure acetonitrile to 30% (v/v)
final concentration, incubation at 50°C for 1 h, and then separation with
50 mM NH4HCO3 containing up to 10% (v/v) acetonitrile. (C) Ureaand (D) guanidine-denaturing conditions involved sample solubilization in 8 M urea or 6 mol/L guanidine 䡠 HCl prepared in 50 mM
NH4HCO3 buffer, incubation at 37°C for 30 min, followed by separation with 6.5 mol/L urea, 50 mM NH4HCO3 eluent. Reducing agents
for cleavage of disulfide bonds were not applied to assess the importance of noncovalent interactions.
Peak detection was monitored with ELSD or absorbance and fluorescence detectors connected in series. The first two conditions (A, B)
are compatible with the ELSD, whereas the latter two conditions (C, D)
are not. The five protein standards and vitamin B12 were subject to all
four treatments for both calibration and assessment of denaturation
effects. Commercially available RuBisCo required an initial and simple
purification step that used SEC with 50 mM NH4HCO3 to collect the
high molecular mass protein (550 kDa), followed by concentration via
lyophilization. All samples were centrifuged (16,000 g, 10 min) to
pellet any unsolubilized protein before injection. Data were not acquired beyond the lowest molecular mass marker (vitamin B12) under
denaturing conditions because of potential fluorescence and absorbance
contributions from buffer components. SEC calibration under denaturing conditions is approximate.
1469
2.5. Proteolytic Digestions
To test whether extracted proteins are accessible to proteolytic attack
even in an aggregated state, samples were incubated under native
conditions with trypsin, which is specific at cleaving the peptide bond
following arginine and lysine residues, or with proteinase-K, a nonspecific enzyme (Creighton, 1993; Copeland, 1994). SEC was then
used to examine the Mr distribution of substrates and any products.
Protein samples were not denatured and reduced before incubation
because a complete digest for peptide mapping purposes was not the
intent (Copeland, 1994), and the strongest denaturation agents would
not be compatible with ELSD.
To avoid contamination of the samples with the proteases for SEC,
insoluble trypsin or proteinase-K attached to cross-linked beaded agarose was prepared and used according to manufacturer guidelines.
Briefly, the insoluble protease in the form of a suspension was gently
mixed, and an aliquot was transferred to a microcentrifuge tube. The
suspension was centrifuged (80 g, 3 s), and the supernatant was
discarded. The packed gel was resuspended and washed with 50 mM
NH4HCO3 a total of six times. A known volume of the working
suspension was used for each 200 ␮L of sample (⬃500 ␮g to 5 mg total
protein) prepared in 50 mM NH4HCO3 buffer, equivalent to 0.35 and
0.2 enzyme units per sample for the trypsin and proteinase-K digestions, respectively. As positive controls, standard proteins (bovine
serum albumin and RuBisCo) were prepared at a concentration of 1
mg/mL in NH4HCO3 buffer, and 200 ␮L (200 ␮g) of each was formed
into aliquots in microcentrifuge tubes with the protease. A negative
control consisted of buffer and protease alone. Incubations were performed at 37°C for 18 h, with mixing. These digests were extended
beyond the 2- to 4-h incubations typically performed for denatured
proteins (Copeland, 1994) to ensure adequate time for substrate– enzyme interaction. After incubation, samples were centrifuged (80 ⫻ g,
3 s) to pellet the insoluble protease, and the supernatants were transferred to new microcentrifuge tubes for centrifugation at higher gforce
(16,000 ⫻ g, 10 min) before injection of soluble material onto the SEC
column. Elution and detection were performed as described above for
SEC and ELSD, with 10% acetonitrile in 50 mM NH4HCO3 used as
eluent. Addition of acetonitrile was intended to minimize interaction of
cleavage products with the stationary phase, in particular for the more
effective separation of hydrophobic peptides (Copeland, 1994; Hedlund
et al., 1998).
3. RESULTS
3.1. Effects of Sample Processing on SEC Protein Elution
Very high Mr protein aggregates eluting in the void volume
(Vo ⬃ 5 mL) of the SEC column appeared to be characteristic
of fresh and lyophilized algal cells as well as both logarithmicand late-stationary-phase cells (Fig. 1). Similar protein elution
profiles were observed for algal cells subjected to NaOH extraction (Figs. 1A,B) and to the trichloroacetic acid/acetone
extractions with solubilization in urea (Fig. 1C). Compared
with the elution profile of algal proteins solubilized in native
NH4HCO3 buffer (Fig. 1D), the bulk of the proteins solubilized
with NaOH or urea eluted slightly earlier. Better separations
were achieved for the NH4HCO3 elution conditions. The earlier
elution of proteins for the NaOH and urea conditions may be
attributed to the potential denaturing effects of urea and the
high pH of NaOH, which could result in an increase in the
hydrodynamic volumes of proteins. At high pH, all the acidic
groups should be dissociated (with a zero or negative charge).
The net negative charge on proteins will result in intramolecular electrostatic repulsion and will favor an extended conformation (Creighton, 1993). Very low Mr material eluting near
the total eluent volume (Vt ⬃ 15 mL) of the column was
observed for the NaOH extracts but not the trichloroacetic acid
extracts, consistent with the initial molecular mass fractionation
1470
R. T. Nguyen and H. R. Harvey
Fig. 2. Calibration curve for proteins and vitamin B12 separated by
SEC under native conditions (NH4HCO3 buffer) and detected by evaporative light scattering. Some protein aggregates elute in Vo; the column exclusion limit is 1,500,000 Da.
Fig. 1. SEC chromatograms of proteins from fresh or lyophilized T.
weissflogii by use of various extraction protocols. (A) Logarithmicphase cells or (B) late-stationary-phase, lyophilized cells extracted with
0.1 N NaOH. Late-stationary-phase, lyophilized cells extracted with
10% (w/v) trichloroacetic acid in acetone (containing 0.1% ␤-mercaptoethanol), then acetone, followed by protein solubilization in (C) 8
mol/L urea, 50 mM NH4HCO3 , and (D) 50 mM NH4HCO3. The void
volume (Vo ⬃ 5 mL) and total buffer volume (Vt ⬃ 14 mL) of the
column are indicated above the respective retention times.
induced by the acid (i.e., ⱗ2000-Da material is not retained in
the protein concentrate).
3.2. ELSD for Protein Analysis and Comparison to
Fluorescence
Although other light-scattering detectors have been used for
protein analysis (e.g., Wen et al., 1996), the application of
ELSD has been mainly limited to carbohydrates, amino acids,
lipids, and synthetic polymers. The effectiveness of ELSD for
protein and small biomolecule detection is evident in its Mr
calibration curve for the SEC column (Fig. 2) and in its ability
to detect nonfluorophores such as vitamin B12 (Table 1). High
molecular mass, multisubunit proteins (from 158 – 670 kDa)
such as thyroglobulin, RuBisCo, and immunoglobulin G exhibited similar ELSD responses of (8 ⫾ 1) ⫻ 104, (5 ⫾ 3) ⫻
104, and (2.5 ⫾ 0.7) ⫻ 104 ␮V 䡠 s/␮g, respectively (Table 1).
These responses were lower than those of the lower molecular
mass biomolecules (from 1.35– 44 kDa) such as ovalbumin,
myoglobin, and vitamin B12, which had similar ELSD responses of (2.5 ⫾ 0.8) ⫻ 105, (2.6 ⫾ 1.0) ⫻ 105, and (4.0 ⫾
0.4) ⫻ 105 ␮V 䡠 s/␮g, respectively (Table 1). Although commonly referred to as an universal mass detector, the ELSD was
biased toward the lower molecular mass biomolecules of the
calibrant mixture. Different responses between these two size
groupings might be attributed to the relatively low resolution
SEC separations. Nonvolatile salts found in some of the commercially prepared protein standards were observed to scatter
light, as evident by chromatographic spikes near Vt, but these
were easy to identify on the basis of a lack of absorbance at 280
nm. Despite the greater precision and up to 1000-fold higher
sensitivity than ELSD, tryptophan fluorescence did not show a
direct relationship to the tryptophan content of known proteins
(Table 1).
3.3. Glucose-Derived Protein–Protein Cross-Links
RuBisCo standards treated with PTB did not exhibit significantly higher BCA assay absorbance responses relative to
controls over a wide concentration range (Table 2). In addition,
no significant differences were observed between calibration
curves for RuBisCo-spiked sediments with and without PTB, or
between the protein recovery factors (difference of y-axis intercepts; Table 2) for treated and untreated RuBisCo as estimated by OPA-HPLC analysis. Without the appropriate standard curve or recovery factor applied to the control or to
treatments, a significant effect of PTB would have been indicated where none existed. PTB was not used on late-stationaryphase diatom cells or on diatom detritus because protein could
be extracted readily from these samples with a simple alkaline
(0.1 N NaOH) extraction protocol (Fig. 3A).
On the basis of the calibration results and on the BCA assay
measure of total “protein,” a significantly higher, albeit small,
release of material for PTB-treated surficial and 4-kyr-old sediments was found (Fig. 3B). BCA assay estimates indicate an 8
and 42% increase in protein recovery for the surficial and
4-kyr-old sediments, respectively, on PTB treatment. Amino
acid analysis (Fig. 3C), although in agreement with total protein estimates for surficial sediments (14% increase), indicates
that the higher BCA assay estimate for the older sediments after
PTB treatment did not represent additional released protein.
The cause of the increase for the 4-kyr-old sediments on the
basis of the colorimetric assay is unknown but could be due to
liberation of sugars that subsequently interfered with the assay.
Protein in marine systems
1471
Table 1. Physicochemical characteristics, and evaporative light scattering detection (ELSD) and fluorescence response factors of pure proteins and
of vitamin B12. Standards were separated under native conditions (50 mM NH4HCO3) by SEC. Response factors (peak area normalized to
micrograms) are mean ⫾ 1 standard deviation (SD) of two injections of different amounts (two fold difference) of each biomolecule.
Fluorescencea
(⫻104 ␮V 䡠 s/␮g)
ELSD
(⫻104 ␮V 䡠 s/␮g)
Biomolecule
Molecular
mass (kDa)
Mean
⫾SD
Mean
⫾SD
No. Trp
residuesb
Total no. AA
residuesb
Thyroglobulin
RuBisCo
Immunoglobulin
Ovalbumin
Myoglobin
Vitamin B12
670
550
158
44
17
1.35
8
5
2.5
25
26
40
1
3
0.7
8
10
4
6.4
7.2
2.2
20
2.9
0
0.8
0.1
0.2
1
0.4
0
78
96
10
3
2
0
5500
4768
660
385
153
0
a
Excitation and emission wavelengths of 280 and 340 nm, respectively; least sensitive photo multiplier settings.
The number of tryptophan (Trp) residues and total number of amino acid residues were obtained for each protein from the SWISSPROT data bank.
Numbers are not available for the entire IgG molecule but are presented for the human IgG heavy chain constant region.
b
Regardless of whether PTB was included in the phosphate
buffer, protein extraction efficiency was high for diatom cells,
diatom detritus, and surficial sediments, with 80 to 90% of the
total solid-phase proteinaceous material removable by buffer
(Fig. 3). Solubilization was lower in the 9.7-m-deep sediments,
with only 14% of proteinaceous material being extracted.
PTB had no significant effect on the relative distribution of
amino acids in RuBisCo, a nonglycosylated protein (Table 3).
However, significant differences (unpaired t-test; p ⬍ 0.05)
were observed between treatment and control for a few amino
acids in the buffer extracted material from Mangrove Lake
sediments. For surficial sediments treated with PTB, mole
percentage (mol%) values for aspartic acid and serine were
lower than those for untreated sediments, whereas those for
tyrosine was higher. Older sediments treated with PTB contained only threonine in abundance significantly higher than the
control.
When surficial and 9.7-m-deep sediments are compared
within either control or treatment groups, several differences in
amino acid composition are observed (Table 3). Most striking
is that the glycine content (20 –24 mol%) in the protein extract
from the deepest sediments was significantly enriched (p ⬍
0.01) compared with surficial sediments (14 mol%). No glycine
enrichment was observed, however, in the total hydrolyzable
amino acid pool down core (Nguyen and Harvey, 1998). Finally, the amino acids histidine, threonine, arginine, tyrosine,
valine, isoleucine, and valine were significantly (p ⬍ 0.05)
depleted in the protein from the deepest sediments.
SEC with absorbance and tryptophan-like fluorescence measurements indicate the preservation of proteinaceous material
of Mr 1000 to 1,500,000 in Mangrove Lake sediments (Fig. 4).
Soluble proteinaceous aggregates eluting in Vo would have a
Mr ⲏ 1,000,000 and were a significant contributor to the total
chromophoric response of the sediment samples. Although
tryptophan-like fluorescence for these aggregates in the 9.7-mdeep sediments was low, tyrosine-like fluorescence (data not
shown) was substantial and would explain the strong absorbance at 280 nm due to contributions from both aromatic amino
acids. Differences in protein Mr distribution for PTB treated vs.
untreated samples were observed only for surficial sediments
(Fig. 4). The significant increase (p ⬍ 0.05) in absorbance and
fluorescence for the protein aggregates in the recent sediments
after PTB treatment might be attributed to an increased extraction of tyrosine-containing (see Table 3) and tryptophan-containing protein. Tryptophan could not be quantified by amino
acid analysis because it is degraded during HCl hydrolysis.
Table 2. Linear regression parameters for control and PTB treatments at different amounts (␮g) of RuBisCo. Equation, y ⫽ a ⫹ bx, where y, a,
b, and x are the dependent variable, y-intercept, regression coefficient, and independent variable, respectively. p ⬍ 0.001 for all regression
coefficients.a
Treatment
Protein
Protein
Protein
Protein
Protein
Protein
(control)
⫹ PTB
⫹ sediment (control)d
⫹ sediment ⫹ PTBd
(control)
⫹ PTB
Detection
method
n
y units
a ⫾ 95% CL
(y units)
b ⫾ 95% CL
(y units/␮g)
R2
BCA assay
BCA assay
BCA assay
BCA assay
OPA-HPLC
OPA-HPLC
14
12
8
8
5
5
AU
AU
AU/mg sediment
AU/mg sediment
␮g protein recovered
␮g protein recovered
(⫺2.0 ⫾ 1.7) ⫻ 10⫺2b
(⫺1.5 ⫾ 1.8) ⫻ 10⫺2b
(1.3 ⫾ 1.9) ⫻ 10⫺2b
(3.0 ⫾ 2.2) ⫻ 10⫺2b
⫺3.8 ⫾ 18.4b
19.9 ⫾ 16.5b
(7.3 ⫾ 0.7) ⫻ 10⫺4c
(8.3 ⫾ 1.1) ⫻ 10⫺4c
(4.4 ⫾ 1.0) ⫻ 10⫺4c
(5.7 ⫾ 1.2) ⫻ 10⫺4c
(4.2 ⫾ 0.9) ⫻ 10⫺1c
(3.9 ⫾ 0.6) ⫻ 10⫺1c
0.98
0.96
0.95
0.96
0.99
0.99
a
PTB, N-phenacylthiazolium bromide; BCA assay, bicinchoninic acid assay; OPA-HPLC, o-phthaldialdehyde high-performance liquid chromatography analysis of protein hydrolyzate; AU, absorbance units; CL, confidence limit.
b
Intercepts are equal for control and PTB treatment based on the test for common intercepts using separate regression fits, at ␣ ⫽ 0.05 (Kleinbaum
et al., 1998).
c
Lines are parallel for control and PTB treatment based on the test for parallelism using separate regression fits, at ␣ ⫽ 0.05 (Kleinbaum et al.,
1998).
d
Protein was mixed with 2.0 to 2.5 mg of 9.7-m-deep sediment from Mangrove Lake.
1472
R. T. Nguyen and H. R. Harvey
conditions (Fig. 6). Late-stationary-phase diatom cells not only
contained proteins of Mr 17,000 to 670,000, but also very high
Mr, multisubunit proteins or protein aggregates eluting in Vo.
This material in the diatom cells was characterized by a strong
ELSD response but had a relatively low fluorescence. In the
diatom detritus and in sediments, little or no lower Mr material,
such as that observed in living cells, could be detected by
ELSD. Instead, high Mr material in the excluded volume predominated.
Unlike PTB, denaturing conditions generally caused dramatic shifts in the Mr distribution of proteinaceous material
from algae, detritus, and sediments. Acetonitrile at 30% final
concentration partially denatured the protein aggregates, with
the greatest shift to lower Mr material observed for the small
quantity of extractable protein in the oldest sediments (Fig. 6).
Acetonitrile concentrations of 5 and 10% final concentration
were tried on the soluble protein aggregates, but 30% provided
effective denaturation that was also compatible with the SEC
column. The hydrophilic calibrant proteins were not soluble
under acetonitrile conditions; calibration was based on the
known void volume and the elution volume of vitamin B12 and
by comparison to the elution of the calibrants under native
conditions. Although the ELSD could not be used with other
denaturing agents, ELSD and absorbance measurements for the
different samples under acetonitrile and urea denaturing conditions, respectively (Fig. 6), were comparable and indicated
similar effects of both denaturants. Denaturation appeared to be
most effective with the 6 mol/L guanidine 䡠 HCl treatment, with
much of the sediment aggregates dissociated into lower Mr
material (Fig. 6). Unlike ELSD or absorbance responses for the
aggregates, fluorescence associated with the high Mr material
exhibited little or no change from native to guanidine 䡠 HCl
conditions.
3.5. Proteolytic Digestions
Fig. 3. Extractable protein from algae, algal detritus, and sediments,
and the effects of PTB. (A) Sodium phosphate buffer extractable
protein (mean ⫾ 1 standard deviation; n ⫽ 2) from diatom cells and
detritus analyzed by BCA colorimetric assay. (B) BCA assay and (C)
OPA-HPLC determined concentrations (mean ⫾ standard error; n ⫽ 3)
of protein extracted from Mangrove Lake sediments by use of sodium
phosphate buffer (control) and that amended with PTB. Dashed lines
represent concentrations of acid-hydrolyzable amino acids from the
solid-phase material and indicate the level of buffer-extractable proteinaceous material. An unpaired t-test was performed for samples at
each depth. Significance levels: *p ⬍ 0.05; **p ⬍ 0.01; ***p ⬍ 0.001.
3.4. Hydrophobic and Other Noncovalent Interactions
Multisubunit proteins are typically associated by various
noncovalent interactions. This was demonstrated when spinach
RuBisCo, a very high molecular mass protein (holoenzyme:
550 kDa), was denatured with 8 mol/L urea into its component
eight large (55 kDa each) and eight small (14 kDa each)
subunits (Fig. 5). Furthermore, some protein aggregates were
part of the commercial preparation, and urea was fairly effective at denaturing these.
As described earlier, very high Mr aggregates were major
contributors to the total protein extracts from sediments on the
basis of absorbance. The quantitative importance of the soluble
protein aggregates in algal and sediment samples was apparent
with ELSD responses of the protein extracts under native
RuBisCo was readily hydrolyzed by trypsin, with no detection of the residual holoenzyme after the 18-h incubation (Fig.
7). Virtually all of the proteins of Mr 17,000 to 670,000 from
the diatom cells were hydrolyzed to lower Mr material. Much of
the protein aggregates of substantial Mr (ⲏ1,000,000) in the
algae, algal detritus, and sediments also were receptive to
proteolytic cleavage, with the bulk of the products between Mr
1000 and 17,000 (Fig. 7). Some material also eluted after Vt,
suggesting noncovalent interactions of that material with the
stationary phase. Proteinase-K digestions produced Mr distributions similar to the trypsin digestions of RuBisCo and protein
extracts from diatom cells and detritus (Fig. 8). Analysis of the
control (proteases in buffer) indicated that small amounts of
low Mr (1000 –17,000) products may be formed. These appear
to be autodigestion products of protease molecules on adjacent
agarose beads over the extended incubation time. Although
some protease autodigestion products might have contributed
to the chromatographic profiles for all samples, the maximum
contribution was ⬍11%, based on peak areas.
4. DISCUSSION
4.1. Role of Noncovalent Interactions
The observation of very high Mr proteins eluting in the void
volume for cells in both logarithmic and late-stationary phases
Protein in marine systems
1473
Table 3. Effect of PTB on the mole percentage composition of RuBisCo and protein isolated from surficial and 9.7-m-deep (4000-yr-old) Mangrove
Lake sediments. Values are the mean and standard error of three to five replicate hydrolyzates.a
RuBisC0
⫹ pTB
(n ⫽ 5)
RuBisCo
control
(n ⫽ 5)
Surficial
sediment
⫹PTB
(n ⫽ 3)
Surficial
sediment,
control
(n ⫽ 3)
9.7-m-deep
sediment
⫹ PTB
(n ⫽ 3)
9.7-m-deep
sediment,
control
(n ⫽ 3)
Amino
acid
Mean
⫾SE
Mean
⫾SE
Mean
⫾SE
Mean
⫾SE
Mean
⫾SE
Mean
⫾SE
Asp
Glu
Ser
His
Gly
Thr
Arg
Ala
Tyr
Met
Val
Phe
Ile
Leu
Lys
BALA
GABA
AABA
9.8
12.2
6.2
2.5
13.1
5.5
4.6
9.7
4.5
0.15
8.0
5.0
4.8
10.1
3.8
—
—
—
0.30
0.35
0.30
0.15
0.48
0.26
0.25
0.60
0.27
0.043
0.26
0.26
0.24
0.36
0.18
—
—
—
9.1
12.4
6.3
1.7
15.3
5.2
4.7
9.4
4.6
0.15
7.5
5.2
4.7
9.8
4.0
—
—
—
0.44
0.14
0.28
0.11
1.1
0.21
0.21
0.46
0.41
0.054
0.23
0.33
0.35
0.61
0.21
—
—
—
9.9b
7.1
4.8b,d
1.3d
14.2d
7.0d
4.0d
9.3
5.4b,d
0.26
7.9d
6.9
6.5d
8.7d
5.4
0.71
0.42
0.20
1.3
1.0
0.78
0.23
0.74
0.38
0.63
1.1
0.27
0.061
0.26
0.35
0.29
0.24
1.4
0.13
0.10
0.053
14.1c
11.1d
7.6c
0.39d
14.3d
6.75d
2.34d
10.0
4.06c,d
0.114
7.62
5.71
5.47
8.05
1.8
0.29
0.13
0.073
0.62
0.045
0.16
0.012
0.30
0.041
0.058
0.20
0.042
0.009
0.049
0.047
0.041
0.028
0.20
0.020
0.010
0.008
11.9
9.1
8.0e
0.24e
20.5e
5.0b,e
1.07e
10.7
3.2e
0.21
6.8e
5.9
4.87e
7.18e
4.5
0.45
0.27
0.14
0.43
0.30
0.17
0.011
0.31
0.10
0.039
0.21
0.11
0.029
0.12
0.17
0.035
0.092
0.70
0.060
0.032
0.022
10.5
9.5e
7.1
0.20e
24.0e
4.1c,e
1.04e
10.6
2.7e
0.21
6.7
6.1
4.8
7.2
4.5
0.44
0.29
0.15
0.61
0.32
0.40
0.017
0.61
0.13
0.038
0.26
0.20
0.022
0.23
0.22
0.19
0.27
0.55
0.052
0.031
0.016
a
Abbreviations for nonprotein amino acids: BALA, ␤-alanine; GABA, ␥-aminobutyric acid; AABA, ␣-aminobutyric acid. A dash indicates not
present. SE, standard error; pTB, N-phenacylthiazolium bromide.
b,c
Mole percentages for an individual amino acid in control and PTB treatment for a particular sediment depth are significantly different (p ⬍ 0.05)
by unpaired t-test.
d,e
Mole percentages for an individual amino acid in surficial and 9.7-m-deep sediments within corresponding treatments are significantly different
(p ⬍ 0.05).
of growth suggests that these protein aggregations can exist
before senescence. Remaining proteins appear to be largely
monomeric on the basis of a comparison of chromatograms for
proteins separated under native and denaturing conditions. The
SEC size distribution of diatom proteins after 6 mol/L guanidine 䡠 HCl treatment is consistent with the distribution observed
by gel electrophoretic methods under denaturing conditions
that showed the predominance of proteins ⬍ Mr 200,000
(Nguyen and Harvey, 1998). The Mr distribution of proteins
found in diatom cells is also similar to that observed for
freshwater green algae during the same phase of growth
(Nguyen and Harvey, in prep.). This is not surprising considering the similar protein amino acid composition of different
algae (Nguyen and Harvey, 1997) and the fact that many of the
same proteins are needed for the various metabolic pathways
and structural roles in algae. The observed Mr distribution of
proteins from intact algae are consistent with the calculated
average Mr (31,700) of polypeptide chains from eukaryotic
cells (Creighton, 1993).
On the basis of separation under native conditions, the very
high Mr proteins in intact cells appear to persist in degraded
material and sediments. Here the phrase “native conditions”
refers to nondenaturing solubilization and SEC elution conditions, as it is unlikely that most proteins in the geosphere would
be expected to retain their original three-dimensional structure
(Collins et al., 1998). Processes such as sorption, for example,
can readily lead to protein unfolding (Taylor et al., 1994). Our
SEC results suggest that proteinaceous material ⲏ Mr
1,000,000 can be preserved in phytodetritus and sediments. A
recent study of Long Island Sound sediments (Pantoja and Lee,
1999) suggests that proteinaceous material ⬎ Mr 100,000 predominates, on the basis of ultrafiltration of NaOH extracts. The
methodology used in that work, however, is limited by the
maximum molecular mass cutoff of the available membranes
and may be conservative. Because protein separations by SEC
or ultrafiltration will depend not only on a protein’s molecular
mass but also on its overall shape—i.e., hydrodynamic radius
or volume (Copeland, 1994; Wen et al., 1996; Kriwacki et al.,
1997)—Mr information must be interpreted with caution. As an
absolute measure of molecular size, multiangle light scattering
confirms that SEC separates mainly according to size (von
Wandruszka et al., 1999).
The very high Mr material observed in algae, detritus, and
sediments might include a variety of proteins or their modified
products. Clp proteases, large enzymes that in higher plants are
located in chloroplasts, have been shown to form heteromeric
complexes of up to Mr 1,700,000 (Sokolenko et al., 1998).
These proteases are found in phytoplankton (Berges and
Falkowski, 1996) and might survive diagenesis. Other multisubunit proteins normally found in algae are additional possibilities. Extracted material could include integral membrane
proteins that are only partially soluble in the aqueous buffers
and therefore would aggregate in solution. A study by Laursen
et al. (1996) suggests that detritus may be selectively enriched
in cell membrane fragments with associated proteins. Because
membrane proteins might remain associated with some membrane fragments, SEC could yield apparent molecular masses
that are higher than the proteins’ actual molecular masses.
1474
R. T. Nguyen and H. R. Harvey
Fig. 4. SEC chromatograms under native (sodium phosphate buffer) conditions for control and PTB-treated Mangrove
Lake sediments. Mr (⫻10⫺3) markers from left to right: 1500, 670, 158, 44, 17, and 1.35. Asterisks indicate significant
difference (p ⬍ 0.05) in absorbance/fluorescence for protein aggregates in control and PTB-treated samples on the basis of
average peak area of three replicates.
Hydrophobic proteins such as lipases have been shown to
exhibit a strong tendency to aggregate (e.g., Rúa et al., 1997).
Besides algal proteins, bacterial S-layer (outer coat) proteins,
which are deficient in methionine, have been suggested as
potential contributors to the slowly hydrolyzable protein fraction in sediments (Mayer et al., 1995). These bacterial proteins
might also be a contributor to Mangrove Lake sediments on the
basis of the low contribution of methionine (⬍0.26 mol%;
Table 3).
Additional contributions to the very high Mr material in the
void volume might be from the aggregation of partially unfolded proteins with each other or with other cellular components during diagenesis. The thermodynamic stability to the
native conformation of a protein relative to the unfolded state is
surprisingly small (Copeland, 1994). Consequently, proteins
can denature in solution over time, leading to the formation of
aggregates. One study indicates that partially denatured proteins are more likely to associate with liposomal lipid bilayers
(Yoshimoto et al., 1998). Partial unfolding might be induced by
changes in ionic strength upon disruption of the cell membrane
during cellular death. This speculation is based on the observation that aggregation of pure proteins may occur in solutions
of different ionic strength (Magiera and Krull, 1992; Rúa et al.,
1997).
As independent evidence for aggregate formation by association of hydrophobic proteins, we stored the green-fluorescent
protein, a conformationally stable protein from Pacific Northwest jellyfish Aequorea victoria, in solution at room temperature for several months and observed aggregation to occur.
Interestingly, 30% acetonitrile had no denaturing effect on the
green-fluorescent protein aggregates, and 8 mol/L urea had
only a small effect, indicating the truly hydrophobic nature of
the material (data not shown). Whereas RuBisCo is readily
denatured with a weak chaotropic agent, urea, extensive denaturation is observed with the “aggregates” from algae, detritus,
and sediments only by use of guanidine 䡠 HCl. Urea and
guanidine 䡠 HCl increase the solubilities of both polar and nonpolar amino acids, disrupting hydrogen bonding and hydrophobic interactions (Creighton, 1993). Whatever the composition
of the void volume material, these results suggest that much of
the proteinaceous material in phytodetritus and sediments is
associated by strong noncovalent interactions.
During the trichloroacetic acid/acetone extractions, proteins
in lyophilized samples were insoluble, whereas pigments, many
lipids, and small peptides were solubilized and removed. Proteins were then solubilized with NH4HCO3, and any insoluble
precipitates were removed at the high g force of centrifugation.
Although the trichloroacetic acid procedure is an inherently
Protein in marine systems
Fig. 5. SEC chromatograms under native (NH4HCO3 buffer) and
urea-denaturing conditions for RuBisCo. RL, RuBisCo large subunits;
RS, RuBisCo small subunits; a, very high Mr protein aggregates.
denaturing one (inducing precipitation) and may not be fully
reversible (Copeland, 1994), most of the native buffer solubilized proteins from the intact algae do not appear as very high
Mr aggregates that elute in the void volume (Fig. 1D). Furthermore, the elution appears consistent with the size distribution of
proteins in cells (Creighton, 1993). This fact, along with the
ability for rapid purification and improved SEC separation,
provided justification for use of this protocol to process all
samples for native SEC analysis. It is possible that part of the
proteinaceous material eluting in Vo was material that aggregated as a result of the isolation procedure, but even the mildest
extractions and purification treatments occasionally modify the
structure of proteins. If this is the case, then we can conclude
that detrital proteins are highly prone to aggregations, whereas
only a small fraction of proteins in intact algae are reactive.
The Mr distribution of sedimentary proteins observed by
denaturing SEC is consistent with that determined by denaturing gel electrophoresis (Nguyen and Harvey, 1998). In this
previous study, mainly unfolded, sedimentary proteins ⬍ Mr
200,000 persisted during the diagenetic process. The lack of
many discrete proteins observed by two-dimensional gel electrophoresis (Nguyen and Harvey, 1998) suggests that the proteinaceous materials could be mixtures of highly cross-linked
material and a few discrete proteins that are noncovalently
associated. Unless proteins are very soluble in a particular
chosen buffer, however, even two-dimensional gel electrophoresis, generally considered the best method for resolving
complex protein mixtures, cannot achieve complete resolution.
Thus, the few discrete protein species seen does not preclude
the possibility that more exist.
Furthermore, the electrophoretic method employed would
not resolve nondisulfide, covalently linked proteinaceous ma-
1475
terial of substantial Mr (⬎200,000), as this material would not
be able to effectively penetrate the pores of a 15% polyacrylamide gel matrix used. SEC results with guanidine 䡠 HCl
suggest that some aggregates may not be completely dissociated even under the strongest denaturing conditions. If the
anionic surfactant sodium dodecyl sulfate, used in the gel
electrophoresis, is as effective as guanidine 䡠 HCl in protein
denaturation, then not all proteinaceous material may have been
detected by the electrophoretic method. Nevertheless, the
present study indicates that the broad Mr range of material with
acidic isoelectric points observed in our previous study results
from the dissociation of proteinaceous aggregates.
Several possibilities may explain the observation that some
aggregates in diatom cells, detritus, and sediments were recalcitrant to even the strongest denaturant. Incomplete denaturation could be partly due to a protein concentration effect
because aggregates are more easily dissociated in denaturing
solutions containing low amounts of protein. Further dilution
(20-fold) of the aggregates in guanidine 䡠 HCl resulted in some
additional dissociations (data not shown). Incomplete dissociations might be attributed to cross-linked material that is unaffected by the denaturing agent. Although disulfide bridges
between cysteine residues of different polypeptides do contribute to the structure of some proteins, these linkages likely
would not be important in proteinaceous materials of anoxic
(reducing) sediments. Reducing agents were applied in our gel
electrophoresis (Nguyen and Harvey, 1998) but not in the SEC
experiments.
The similar Mr distributions observed for both methodologies would suggest that noncovalent interactions, rather than
disulfide linkages, are important. Another explanation for the
apparent incomplete aggregate dissociation is that the dissociated material unfolds and has a sufficiently large hydrodynamic
volume to cause its elution in the excluded volume. Unfolding
would result in an increase in the hydrodynamic volume of
monomeric proteins and is observable by SEC (Shalongo et al.,
1993; Sarkar and DasGupta, 1996; Wen et al., 1996; Herbst et
al., 1998). This unfolding and earlier elution would give the
apparent observation that the guanidine 䡠 HCl conditions are not
completely effective at denaturation. For the T. weissflogii
cells, the presence of both aggregates and lower Mr proteins
complicates the interpretation of aggregate dissociation, because elution of material in the void volume under denaturing
conditions may include some undissociated aggregates; lower
Mr but completely unfolded proteins with increased hydrodynamic volumes; or both.
A comparison of the different HPLC detection methods
indicates the use of multiple detectors for protein analysis of
mixtures. Both ELSD and absorbance yielded similar information, but the quantitative importance of the aggregates was fully
realized only with ELSD. SEC results clearly show that fluorescence cannot yield quantitative information for protein mixtures of unknown composition. Protein fluorescence is largely
determined by the number of tyrosine or tryptophan residues
and the location of these residues in a particular protein molecule (Copeland, 1994). In addition, the fluorescence intensity
and emission maximum changes upon protein unfolding and
varies with solvent conditions (Sarkar and DasGupta, 1996;
Determann et al., 1998). Tyrosine fluorescence measurement is
not particularly useful for folded proteins because peptide
1476
R. T. Nguyen and H. R. Harvey
Fig. 6. SEC chromatograms under native (NH4HCO3 buffer) and acetonitrile-, urea-, and guanidine 䡠 HCl-denaturing
conditions for algal proteinaceous material during different stages of decay. Mr (⫻10⫺3) markers from left to right for the
first two conditions monitored by ELSD and fluorescence: 1500, 670, 158, 44, 17, and 1.35. Approximate Mr (⫻10⫺3)
markers from left to right for the latter two conditions monitored by absorbance and fluorescence: 1500 –335, 158, 44, 17,
1.35.
bonds or the presence of tryptophan may quench the fluorescence of tyrosine (Copeland, 1994). For a particular protein, an
increase in unfolding may be deduced by an increase in tyrosine
fluorescence (Shalongo et al., 1993). The absorbance response for
the aggregates extracted from the 9.7-m-deep Mangrove Lake
sediments could be accounted for by a strong tyrosine-like fluorescence, suggesting that the proteinaceous material in the deeper
sediments may be in an highly unfolded state. Regardless of the
limitations of fluorescence measurements for analyzing environmental samples, these measurements do supplement the
absorbance and ELSD measurements in providing information
on the proteinaceous nature of various biomolecules.
4.2. Peptide Linkages in Algal Protein and Sedimentary
Protein Aggregates
The efficient proteolytic digestion of the RuBisCo holoenzyme
to lower Mr peptides could be attributed to trypsin’s potential
access to a high number of cleavage sites, a total of 528 arginine
plus lysine residues (Table 4). Not all sites would be available
immediately, especially because the protein was incubated under
native conditions. Upon cleavage of exposed polypeptide chains
containing arginine and lysine, other sites would be made available over time. For T. weissflogii, it is difficult to estimate the total
number of arginine and lysine residues in various proteins because
there is a wide Mr distribution of proteins in the alga.
Compared with the degradation of RuBisCo, less extensive
hydrolysis of the aggregates from the diatom detritus and from
sediments (72–98% loss to lower Mr material; Table 4) could
be attributed to differences in amino acid composition and in
structural folding. Tightly associated proteins, in general,
would be expected to resist degradation, as many of the peptide
bonds would be protected from enzymatic attack. Local unfolding probably precedes proteolytic cleavage of the polypeptide
chain (Creighton, 1993). Thus, the stability of a protein would
be expected to determine its rate of degradation. The preferential preservation of aggregated or high Mr multisubunit phytoplankton proteins in sediments suggests that tight associations
confer some resistance to enzymatic attack. Assuming that the
aggregates are mainly proteinaceous, there would be a lower
number of arginine plus lysine residues relative to the total
number of amino acid residues in these macromolecules as
compared to RuBisCo (Table 4).
The near-complete digestion (98% loss to lower Mr) of the
aggregates isolated from the 4-kyr-old sediments might be due
to their higher abundance of arginine plus lysine as compared
with that in aggregates from the other degraded samples (Table
4). The extensive cleavage of aggregates by trypsin suggests
that many of the lysine residues have not undergone Schiffbase condensation reactions with sugars, although it is difficult
to ascertain what extent of cleavage could have occurred if
Protein in marine systems
Fig. 7. SEC chromatograms of undigested and trypsin-digested
RuBisCo and algal proteinaceous material from different diagenetic
stages. Digestions were performed under native conditions (NH4HCO3
buffer). Inset: magnified view of very high Mr aggregates in 4-kyr-old
sediments. Mr (⫻10⫺3) markers from left to right: 1500, 670, 158, 44,
17, and 1.35.
trypsin had access to only arginine residues. Although the
lysine residues in the 4000-yr-old, buffer-extractable proteins
are probably not cross-linked, it must be emphasized that only
a small fraction (14%) of the total proteinaceous material in the
oldest sediments is extractable without acid hydrolysis.
The presence of peptide bonds in sediments is indicated by
other studies that have applied proteases to look at enzymatically available protein (Mayer et al., 1986, 1995; Laursen et al.,
1996; Dauwe et al., 1999a,b). The current results, however, are
the first to show preservation of peptide bonds associated with
both very high Mr sedimentary material and with material as
old as 4000 yr in sediments where mineral sorption is limited.
These results are also supported by analysis of 15N nuclear
magnetic resonance data that shows the predominance of nitrogen in the amide linkage down core in Mangrove Lake
sediments (Knicker et al., 1996; Knicker and Hatcher, 1997).
Surprisingly, limited protein hydrolysis can also lead to
protein aggregations. Aggregations may occur as polypeptides
unfold upon initial cleavage, with the newly exposed hydrophobic regions subsequently interacting with hydrophobic regions of other molecules (e.g., Otte et al., 1997). In addition to
cleavage by proteases, protein hydrolysis may be through the
spontaneous deamidation of asparagine and glutamine residues
(Creighton, 1993). A fraction of asparagine residues are converted to isoaspartic acid residues, resulting in the peptide bond
1477
Fig. 8. SEC chromatograms of proteinase-K– digested RuBisCo and
algal proteinaceous material. Digestions were performed under native
conditions (NH4HCO3 buffer). Mr (⫻10⫺3) markers from left to right:
1500, 670, 158, 44, 17, and 1.35.
occurring through the side chain. This can have severe effects
on protein structure and may lead to protein hydrolysis and,
perhaps, to aggregation.
The observed enzymatic hydrolysis of detrital and sedimentary proteins after their extraction seems enigmatic, especially
because minerals are absent or present in very low abundance.
One possibility for this observation is that these proteins are
protected on long time scales by intimate associations with the
detrital organic matrix. The encapsulation hypothesis of
Knicker and Hatcher (1997) proposes that proteins incorporated within the macromolecular matrix forming sedimentary
organic matter are sterically protected from bacterial hydrolysis. Specifically, encapsulation could occur by the hydrophobic
interaction of aliphatic amino acid residues of polypeptides
with paraffinic regions of humic acids (Zang et al., 2000) or by
protection by lipids (Borch and Kirchman, 1998).
4.3. Protein–Protein Cross-Links
We initially hypothesized that AGE cross-linked protein
could explain much of the concentration difference seen between the protein profiles in Mangrove Lake sediments by two
different extraction protocols (Nguyen and Harvey, 1998). We
had observed in sediments that the ⬎2 kDa amino acid fraction
exhibited no significant down core changes in concentration,
whereas the NaOH-soluble fraction decreased by two thirds.
Consistent with our findings, Mayer et al. (1995) observed that
NaOH-extractable protein and enzymatically hydrolyzable
amino acids decrease more rapidly down core than total acid-
1478
R. T. Nguyen and H. R. Harvey
Table 4. Total number of amino acids, arginine and lysine composition, the potential number of trypsin cleavage sites, and trypsin digestibility for
RuBisCo and very high Mr proteinaceous aggregates from diatom detritus and Mangrove Lake sediments. Trypsin cleaves on the C-terminal side of
arginine and lysine residues between Arg-X and Lys-X bonds, where X is any residue other than proline.
Protein
Total no.
residuesa
Mole%
Arg
Mole%
Lysb
No Arg
residuesc
No. Lys
residuesc
Total no.
Arg ⫹ Lys
%
Digestedd
RuBisCo holoenzyme, spinach
Aggregates, S. costatum detritus
Aggregates, surficial sediments
Aggregates, 9.7-m-deep sediments
4,768
⬃13,000
⬃13,000
⬃13,000
5.5
1.5
2.3
1.0
5.5
2.2
1.8
4.5
264
195
300
130
264
290
230
585
528
485
530
715
100
92
72
98
a
Total number of amino acid residues for RuBisCo was determined from the SWISSPROT data bank. Spinach RuBisCo holoenzyme is comprised
of eight identical large and eight identical small subunits. Aggregates isolated from detritus in algal decay experiment and from sediments are assumed
(1) to have an average molecular mass of 1,500,000 Da and (2) to be mostly proteinaceous. The total number of amino acid residues was estimated
on the basis of the average anhydro molecular mass (115 Da) of an amino acid.
b
Composition of RuBisCo was determined from the SWISSPROT data bank. Amino acid composition for aggregates based on OPA-HPLC
analysis of buffer-extractable protein (for example, see Table 3. Mangrove Lake sediment control samples).
c
The number of Arg or Lys residues can be calculated knowing the mole percentage (mole%) and total number of amino acid residues. For
example: no. Arg ⫽ (mole% Arg/100) ⫻ Total no. residues. For the aggregates, the major assumption is that Arg and Lys residues are found
throughout the macromolecule rather than confined within lower molecular mass polypeptides that are aggregated to the rest of the proteinaceous
material.
d
Percentage of original protein degested to lower Mr material is based on the peak area of holoenzyme or aggregate before and after trypin digestion
(see Fig. 7). Amount of aggregate digested in T. weissflogii late-stationary-phase cells is 91%.
hydrolyzable amino acids and total nitrogen in coastal sediments.
The difficulty in extracting proteinaceous material from
older sediment sequences with NaOH has also been observed
for Long Island Sound sediments (Pantoja and Lee, 1999). In
the present study, PTB was used to determine if a particular
type of AGE cross-linking played a role in decreasing the
solubility or bioavailability of proteins down core. The similar
results seen in the deepest sediments of Mangrove Lake with or
without PTB treatment suggest that other cross-linking pathways might be responsible for the concentration difference
between alkaline- and acid-extracted material. Alternatively,
some proteins might be tightly encapsulated in the older sediments and very difficult to extract except by harsh acid or base
hydrolysis conditions.
Unlike total acid– hydrolyzable amino acid compositions that
show little variation down core (Nguyen and Harvey, 1998),
the amino acid compositions of buffer/NaOH-soluble protein
from surficial and 9.7-m-deep sediments varied. These differences appear to result from the lower amount of proteinaceous
material from older sediments that can be extracted. Some of
the amino acids significantly depleted (histidine, tyrosine, isoleucine, and leucine) or enriched (glycine) in buffer-extractable
material from the deep sediments have been proposed as indicators of highly degraded organic matter (Dauwe and Middleburg, 1998; Dauwe et al., 1999b). Interestingly, glycine in the
total acid– hydrolyzed sediments exhibited no down core enrichment (Nguyen and Harvey, 1998), suggesting that the residual material that could not be extracted is glycine depleted.
The enriched glycine content of the buffer-extracted material
may reflect contributions from bacterial peptidoglycan (Keil et
al., in press).
On the basis of our SEC and protein extraction data, soluble
protein aggregates should comprise most of the ⬎2 kDa hydrolyzable amino acids in algal detritus and surficial sediments
but only a small fraction in the deepest sediments. Interestingly,
the anionic surfactant sodium deoxycholate was not able to
extract more protein than the traditionally applied NaOH ex-
traction, averaging 71 and 30% of NaOH values in surficial and
9.7-m-deep sediments, respectively (Fig. 9). The inefficient
buffer, surfactant, and NaOH extraction of proteins from the
older sediments cautions that the Mr distribution determined by
SEC may not include all size classes or types of proteins. The
predominance of material from older sediments eluting in the
void volume does indicate that most buffer-soluble material is
characterized by an high Mr. Although PTB treatment does
increase (albeit small) the amount of protein extracted from
surficial sediments, this appears to result from enhanced protein
solubilization rather than cleavage products. This is supported
by SEC separations after PTB treatment, which observed no
low Mr products but rather an increase in aggregates. Nevertheless, it is possible that some of the aggregates are crosslinked to the complex sedimentary matrix and were released by
PTB.
Our PTB results are consistent with 15N nuclear magnetic
resonance spectroscopic analyses of Knicker et al. (1996),
which indicate that Maillard condensation products are not
prevalent in Mangrove Lake sediments. This implies that the
small shift to higher Mr proteinaceous material observed in our
previous denaturing gel electrophoresis experiments (Nguyen
and Harvey, 1998) may be the result of covalent cross-links that
still elude characterization. For the biomedical community, it
has been difficult to determine the dominant structure of the
cross-linking AGEs that form in vivo because of the structural
heterogeneity of the principal cross-links, the low amount of
individual cross-linking species, and the lability of AGEs to the
employed purification conditions (Vasan et al., 1996). Similar
problems, as well as the greater heterogeneity of the matrix,
may be faced by the geochemical community in its attempts to
characterize sedimentary organic nitrogen.
5. CONCLUSIONS
The application of PTB indicates that glucose-derived protein–protein cross-linking formed via an ␣-dicarbonyl intermediate is a minor pathway for the preservation of proteinaceous
material in sediments. Large aggregates or very high Mr mul-
Protein in marine systems
1479
Fig. 9. Percentage of proteinaceous material as extractable, very high Mr aggregates in algal detritus and down core in
Mangrove Lake sediments. Relative amount was determined from the ratio of NaOH- or surfactant-soluble protein to
HCl-hydrolyzable protein (⬎2000 Da amino acids) in the solid-phase material. The diatom detritus is not from the lake, but
data for it are shown on the same graph for comparative purposes. NaOH (0.5 N), the anionic surfactant sodium
deoxycholate (0.16% w/v), and HCl (6 N) hydrolysis extractions were performed as described by Nguyen and Harvey
(1994, 1998). The material that is not extractable with base or surfactant may represent cross-linked or tightly encapsulated
proteins.
tisubunit proteins are present in intact phytoplankton and appear to be preferentially preserved in sediments. Alternatively,
the proteinaceous material may represent that fraction of organic matter that is highly prone to aggregations. The shift in
Mr of extracted proteins treated with various denaturants indicates the importance of macromolecular aggregation by hydrophobic interactions and hydrogen bonding for preservation.
Long-term preservation of peptide linkages in this very high Mr
material also may be attributed to noncovalent associations of
the aggregates with the detrital, organic matrix (e.g., membrane
fragments and aliphatic cell wall material). An extended preservation time afforded by such macromolecular aggregations or
encapsulation might allow time for covalent cross-linkings to
occur that make the proteinaceous material even more refractory.
Acknowledgments—We thank Antonio Mannino for the degraded S.
costatum sample, Matthew Collins for stimulating discussions, and two
anonymous reviewers for comments that improved the final manuscript. This research was supported by the U.S. National Science
Foundation (OCE-9617892 and OCE-9907069) and the donors of the
Petroleum Research Fund of the American Chemical Society. Contribution No. 3391, University of Maryland Center for Environmental
Science.
Associate editor: R. Summons
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