+ model
ARTICLE IN PRESS
Marine Chemistry xx (2006) xxx – xxx
www.elsevier.com/locate/marchem
Intact protein modification and degradation in
estuarine environments
Lori C. Roth, H. Rodger Harvey *
University of Maryland Center for Environmental Science, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA
Received in revised form 8 September 2005; accepted 11 October 2005
Abstract
Proteins are the principal organic nitrogen-containing compounds of living biomass and by virtue of the readily cleaved amide
bonds are believed to be very labile. Nevertheless, proteins have been found in multiple organic matter pools. Experimental
incubations among three diverse estuarine environments (anoxic Chesapeake Bay, Lower Delaware Bay, and freshwater marsh)
were used to examine the initial stages of protein hydrolysis and associated structural modification and degradation of the model
protein bovine serum albumin (BSA). Size-exclusion chromatography combined with electrospray ionization mass spectrometry
observed multiple proteinaceous products of lower molecular weight formed over the course of the incubation. Products ranged
from 63 to 13 kDa, suggesting initial degradation via sequential hydrolysis. Complete hydrolysis of added protein occurred in all
three environments within 48 h. Amino acid analysis of the intermediate products of degradation suggests that the initial process
(hours) involves the selective removal of polar, charged amino acids by bacterial assemblages present. As degradation of the
protein products continues, other functional groups are lost, leaving the overall amino acid composition of remaining material
closely resembling that seen for the intact protein.
D 2006 Elsevier B.V. All rights reserved.
Keywords: Protein degradation; Amino acids; DOM; Carbon cycling; DON
1. Introduction
Proteins are integral components of the dissolved
nitrogen cycle. Because most nitrogen in plankton (believed to be the biggest source of DOM in marine
waters) is present as protein (Lourenço et al., 1998),
it constitutes a likely starting material for much of the
organic nitrogen present in marine environments. The
dominance of dissolved organic nitrogen in aquatic
environments as the amide form (Knicker and Hatcher,
* Corresponding author. Tel.: +1 410 326 7206; fax: +1 410 326
7341.
E-mail address: harvey@cbl.umces.edu (H.R. Harvey).
1997; McCarthy et al., 1997) lends further support that
proteins or their modified products are one source of
this material.
Although proteins were previously considered labile
in marine environments and therefore not readily preserved (Hollibaugh and Azam, 1983), more recent evidence suggests that preservation of proteinaceous
material in aquatic environments does occur (Nguyen
and Harvey, 1997, 1998; Pantoja et al., 1997). This
includes reports of intact proteins observed in ocean
waters (Tanoue, 1995) and the slow rates of turnover
seen for membrane proteins which might allow preservation (Nagata et al., 1998). The mechanisms suggested
for protein preservation or recycling include abiotic
processes such as condensation reactions which result
0304-4203/$ - see front matter D 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.marchem.2005.10.025
MARCHE-02312; No of Pages 13
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
in reduced bioavailability (Hedges, 1978; Nagata and
Kirchman, 1997) and sorption to mineral surfaces
which impede microbial attack (Hedges and Hare,
1987; Keil et al., 1994; Kirchman et al., 1989; Lee
and Ruckenstein, 1988). Additional mechanisms, including hydrophobic interactions (Nguyen and Harvey,
2001) and encapsulation (Knicker and Hatcher, 1997),
allow for the stabilization of proteinaceous material
over longer timescales. In all these processes, however,
there remains a poor understanding of the first steps in
protein recycling, the products produced, and their
longevity in the environment.
It is a matter of debate whether intermediates
created during bacterial degradation of protein can
be found in marine environments. Hollibaugh and
Azam (1983) did not find intermediate peptides
during the degradation of BSA; however, Keil and
Kirchman (1991a) and Pantoja and Lee (1999) did
observe what they considered intermediates during
protein degradation based on the presence of high
molecular weight amino acids. In addition, dissolved
proteins have been observed in a range of natural
waters (Nguyen and Harvey, 1998; Tanoue, 1995;
Tanoue et al., 1996), suggesting that immediate hydrolysis is not universal. The major focus of this
study is the examination of the degradation and
potential modification of protein mediated by microbial processes. We investigated the degradation
mechanism of dissolved protein and modification
as evidenced by structural changes in a model protein in the presence of natural microbial communities. Experimental incubations were used to provide
a comparison of the protein degradation and structural characteristics of intermediate peptide products
during the degradation process. Molecular weight
changes of intact products were followed over time
while amino acid analysis was used to determine if
alterations in molecular weight were accompanied by
changes in major amino acid distribution. The goal
was to examine the initial process of protein degradation by natural microbial consortia and formation
of early degradation products which might contribute
to the dissolved organic nitrogen pool in aquatic
systems.
2. Materials and methods
2.1. Protein degradation incubations
Field sampling took place from August 27 to 31,
2002, at two locations in the Delaware Bay system
characterized by differing physiochemical characteris-
tics. The Marsh site is a freshwater marsh (2) and is
heavily influenced by tidal cycles with significant
amounts of humic and organic material. The Delaware
Bay site is near the mouth of the estuary and characterized by high salinity (31.5) and high productivity
(Sharp et al., 1982; Pennock and Sharp, 1986). A third
site included the deep channel of the nearby Chesapeake Bay which encounters seasonal anoxia, with a
salinity of 15. Anoxic waters were present within 3 m
of the surface during the time of sampling (G. Luther,
unpublished results) and maintained under anoxic conditions throughout the incubation period. Additional
incubations used water collected from the lower
Patuxent River (salinity 10.5) near the entrance to
the mesohaline portion of the Chesapeake Bay. At
each site, one 20 L carboy was used to collect water
via pumping from 1 m depth (or 10 m in anoxic
waters) which was filtered through a 0.2 Am polycarbonate filter to remove all particles including phytoplankton and bacteria. A 10% (v/v) inoculum of water
prefiltered through a 3 Am filter containing the natural
bacterial community was added to the 0.2 Am filtered
water. Bovine serum albumin (15 nM L 1) was added
to the carboy containing the natural bacterial assemblage. The experimental incubations were sampled at
12, 24, 36, and 48 h, with aliquots removed and
frozen at 70 8C until concentration and analysis.
An aliquot of the 0.1 Am filtered surface water
from the Patuxent River was used as an abiotic
control to investigate the potential for short-term
protein modification and was sampled at 0 and 92
h. From each sample, 60 mL was removed and
processed as described below. For parallel experiments, Patuxent River water was collected at 0, 6,
12, 24, 36, 48, and 92 h and then processed as
described below.
2.2. Protein isolation
Dissolved protein in incubation and control waters
was isolated and concentrated using ultrafiltration with
centrifugal filters containing a nominal 5 kDa membrane (Millipore). Each aliquot was initially filtered
through a 0.45 Am PDVP membrane filter (Gelman
Sciences) followed by concentration. The samples
were centrifuged (15,000g) at room temperature to
reduce the volume to 150 AL. The retained concentrate
was then rinsed with 2 mL of 0.16% sodium deoxycholate to minimize absorption to the filter membrane followed by two rinses of 2 mL Nanopure
water to remove excess deoxycholate. The samples
was transferred using 350 AL of 50 mM NH4CO3
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
buffer (pH 7.0) amended with guanidined HCl (3 M
final concentration) to minimize protein aggregation
and stored at 70 8C until analysis.
2.3. Quantification and separation of proteins and
peptides
Size-exclusion chromatography was performed at
room temperature under native conditions, (i.e. mobile
phase without a denaturing agent) using a Superdex
200 HR column (30 cm 10 mm, Amersham Pharmacia) operating with 50 mM NH4HCO3 mobile phase at
0.5 mL min 1. Subsamples were injected and elution
was monitored by fluorescence (k ex = 280 nm; k em =
340 nm). Peaks of interest were collected by fraction
collection and stored at 70 8C until prepared for
further analysis. The performance of the column and
molecular weight calibration was constructed using a
set of known molecular weight proteins including
aprotinin (65 kDa), cytochrome c (12.4 kDa), carbonic
anhydrase (29 kDa), bovine serum albumin (67 kDa),
and blue dextran (2000 kDa-void volume) and a premixed gel permeation chromatography standard including thyroglobulin (670 kDa), gamma globulin
(158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa),
and vitamin B12 (1.35 kDa). A calibration curve of these
molecular weight standards was used to estimate molecular weights of unknowns based on retention time
and repeated after each sample suite. To quantify BSA
and its potential products, known concentrations of BSA
were injected in equal volumes to develop a calibration
curve used to estimate protein concentrations observed
(Fig. 1). Molecular weight resolution of the column and
flow rates used in the present study limited fraction
collection to a minimum of 0.5 minute peak widths for
amino acid analysis, equivalent to ~25 kDa based on
protein calibrants.
BSA concentration (µM)
0.60
y = 1.42x + 3.65
0.45
R2= 0.965
0.30
0.15
0
0
5
10
Peak Area
15
20
25
(x103)
Fig. 1. Calibration curve for pure BSA verses fluorescence peak area
used to determine unknown protein concentrations.
3
2.4. Molecular weight analysis by ESI-LC-MS
Mass determination measurements were performed
using the Agilent 1100 ion trap mass spectrometer
with an electrospray ionization interface. Concentrated
protein samples (752 nM) were infused at 400 AL
min 1, using a mobile phase of 50 mM NH4HCO3
with 0.1% formic acid. The instrument was first tuned
to get the optimal MS conditions for protein
responses. The MS conditions used for all protein
infusions are as follows: capillary voltage, 150–180
V; trap drive, 75–80 V; dry gas temperature, 350 8C;
nebulizer gas pressure, 15.0 psi. The mass spectra
were registered in normal scan mode (m / z, 200–
2000; scan time, 5 scans s 1, maximum accumulation
time, 50 ms, ICC target, 30,000). As the protein was
being infused, the instrument was tuned for final
optimization parameters to obtain the highest
responses for each protein product examined. Because
proteins are multiple-charged compounds, they acquire
a range of charge states yielding a series of masses for
an individual compound. Deconvolution software
(Agilent technologies) allowed statistical transform of
the envelope of multiple-charged products into a singly charged parent spectrum for molecular weight
determination.
2.5. Amino acid analysis
To provide additional information on amino acid
composition of observed degradation products, total
amino acid analysis was conducted on isolated products using methods previously described (Mannino
and Harvey, 2000. Briefly, individual samples were
transferred to a 4 mL amber vial, a known amount
(68.9 AM) of g-methylleucine added as an internal
standard, and then dried with nitrogen gas. The samples were then redissolved in 0.5 mL of sequanal
grade HCl (Sigma Chemical Co), capped under nitrogen, incubated at 150 8C for 2 h. Acid was removed
from hydrolyzed samples under a gentle stream of
nitrogen at 45 8C to approach dryness. The samples
were subsequently derivatized to trifluoracyl isopropyl
esters and analyzed by gas chromatography and gas
chromatography/mass spectrometry (GC/MS) as described by (Silfer et al., 1991). Amino acid concentrations were determined based on the internal
standard and an external standard commercial mix
of the amino acids (Sigma) used for determination
of yields. Variable responses in individual amino
acids were corrected by comparison of peak areas
of each amino acid to g-methylleucine and calcula-
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
Protein concentration (µg/ml)
4
Delaware Bay
that sample processing did not alter amino acid composition. The average (n = 5) coefficient of variation
for individual amino acids between these two
approaches was F2.3%.
Chesapeake Bay
Marsh
1
0.5
3. Results
0
0
20
40
3.1. Comparison of dissolved protein hydrolysis among
environments
60
Time (hrs)
Fig. 2. Time course of degradation for dissolved protein determined
from ultrafiltered (N5 kDa) concentrates in three different natural
environments.
The anoxic Chesapeake Bay, lower Delaware Bay
and freshwater marsh incubations all showed a rapid
decrease in BSA concentration from 0 to 24 h, with
most rapid loss seen in freshwater marsh waters (Fig.
2). By 24 h, BSA was not detected in unconcentrated
marsh incubations and present only in trace amounts at
tion of recoveries. Replicate BSA samples were also
dissolved and concentrated using ultrafiltration and
compared to direct analysis of intact BSA to ensure
Chesapeake Bay
Delaware Bay
A
Marsh
C
B
Fluorescence (λex = 280 nm; λem= 340 nm)
12 hr
24 hr
36 hr
48 hr
10
20
30
40
Elution time (min)
All
0 hr
10
10
20
30
40
Elution time (min)
10
20
30
40
Elution time (min)
Abiotic
92 hours.
20
30
40
Elution time (min)
10
20
30
40
Elution time (min)
Fig. 3. Size-exclusion chromatograms of concentrated dissolved proteins and degradation products present during BSA degradation in the three
different aquatic environments: a) Chesapeake Bay b) lower Delaware Bay and c) Freshwater Marsh. Abiotic controls after 92 h are shown as a
separate inset. All three sites showed identical distributions of protein at time 0 with one example shown as a separate inset for illustration.
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3.2. ESI MS analysis
An additional incubation using Patuxent River estuarine water was used to examine the potential for more
rapid changes than seen by 24 h for all three sites and
provide adequate material for detailed analysis. Incubation procedures were identical, but larger sample
volumes allowed increased sample amounts required
for preparative SEC followed by ESI MS analysis.
Mass determination for concentrated protein products
was performed following size exclusion chromatography (SEC). The addition of BSA to incubation waters
resulted in a rapid appearance of a lower molecular
weight product, seen as a mixture of BSA (66.2 kDa)
plus a 64.8 kDa product within minutes of addition
(Fig. 4). This mass shift corresponds to an average
loss of approximately three amino acids from BSA.
Within 6 h, a subset of lower molecular weight products
(average mass of 63.3 kDa) was also observed,
corresponding to a loss of approximately eleven
amino acids. Intermediate products were in the same
range with slight shifts in molecular weights at 12 h. By
12 h an additional smaller intermediate product was
observed at 45.1 kDa ( 142 amino acids). There were
also shifts in molecular weights from BSA (66.3, 1
amino acid and 57.7 kDa, 58 amino acids). After 24
hours, the largest peak observed had a molecular weight
of 57.9 kDa ( 57 amino acids). Interestingly, an additional hydrolysis product was seen at 12 h (45.1 kDa)
and observed through 24 h with similar intensity. The
0 hr
66,194 Da
64,802 Da
(-11aa)
6 hr
66,379 Da
27.287
Relative Abundance
36 h in the Chesapeake Bay and lower Delaware Bay
incubations. Despite the rapid recycling of dissolved
protein by the microbial community, an examination of
the molecular weight distributions of sample concentrates found a variable pattern of initial products formed
among the three environments (Fig. 3). In all three
environments, both higher and lower molecular weight
products (relative to BSA) were seen by 12 h, with
distributions dependent upon time and site. Incubations
in anoxic Chesapeake Bay waters (Fig. 3a) showed a
consistent loss throughout the incubation period, with a
rapid loss of BSA accompanied by the appearance of
multiple products through 24 h. By 36 h most were lost,
and only small amounts of high molecular weight
products remained at 48 h. In the lower Delaware
Bay site (Fig. 3b), modified products were observed
at 12 h ranging from 10 to N800 kDa in size. The
distribution of modified products changed little at
24 h, with a substantial decrease in proteinaceous material by 36 h. Only the largest molecular weight masses
which might represent aggregated material remained in
the incubation at 48 h.
In contrast, the freshwater marsh incubation differed
significantly from the other sites (Fig. 3c) in that high
molecular weight aggregates or modified products
dominated the degradation products seen. As with incubations at other sites, a number of products were observed at 12 h ranging from 17 to 500 kDa with high
molecular weight material dominant. While most products were subsequently lost from marsh waters by 24
h, a high molecular weight (N 1000 kDa) fraction
remained through 36 h. Unlike either Chesapeake or
Delaware incubations, marsh incubations showed little
evidence of remaining protein or its modified products
at 48 h.
In the control (i.e. abiotic) incubation, BSA was the
dominant peak with only small amounts of high molecular material (likely BSA dimers) also seen (Fig. 3,
92 h abiotic inset), confirming that biotic processes
were responsible for the rapid changes seen in the
bacterized incubations.
5
12 hr
63,339 Da
(-20aa)
57,650 Da
(-58aa)
45,117 Da
(-142aa)
24 hr
57,893
(-57aa)
45,117 Da
(-142aa)
10
20
30
40
Elution time (min)
Fig. 4. Size exclusion chromatography of dissolved protein (BSA)
degradation in Patuxent River waters. The shaded boxes indicate the
peaks collected and further analyzed by ESI MS. The dominant mass
of the peak is shown (in Da) and calculated by deconvolution. The
second value estimates the number of amino acids lost from the
original protein to achieve the calculated mass. Chromatograms are
not to the same scale to allow degradation products to be viewed.
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
longevity of this product in the incubation while others
showed rapid loss indicates that its further degradation
may be at a slower rate than its initial formation.
3.3. Amino acid analysis
Previous work has observed that total amino
acid composition generally remains invariant during
organic matter degradation (Henrichs and Farrington,
1987; Nguyen and Harvey, 1994a, 1997; Siezen and
Mague, 1978). To examine if this holds true during
the initial stages of protein hydrolysis, the major
amino acid composition of the total high molecular
weight dissolved fraction (N 5 kDa–0.45 Am) amended
by BSA and isolated products observed during short
terms exposure to natural microbial assemblages were
compared.
3.3.1. Total amino acids in dissolved protein
Among individual amino acids, the modified products seen in sample concentrates showed only small
shifts in composition over time (Table 1). To more
easily compare functional changes in the products observed, individual amino acids were grouped by side
chain functionality and summed. Using this approach,
the model protein BSA contains 42% charged, polar
amino acids, 51% nonpolar amino acids, and 7% uncharged, polar amino acids (Fig. 5). Within 1 h of the
addition of BSA to the marsh waters, the distribution of
major amino acids comprised charged, polar amino
acids (68%), followed by nonpolar amino acids (30%)
and uncharged, polar amino acids (2%) (Fig. 5-top
panel). By 48 h when the incubation was halted, the
amino acid distribution had shifted to 71% nonpolar,
24% charged polar amino acids and 5% uncharged
polar amino acids. For the overall incubation, there
was an increase among major nonpolar amino acids
and a decrease in those with polar, charged side chains;
uncharged, polar amino acids remained unchanged.
Among individual nonpolar amino acids, glycine and
alanine increased significantly during the incubation,
while valine, phenylalanine, leucine, proline, and isoleucine remained unchanged. Although leucine did not
change in molar contribution through the incubation, it
was lower than that seen in BSA present in abiotic
controls. The decrease in amino acids with the polar
charged side chain appeared mainly due to a large
decrease in aspartic acid; lysine remained largely unchanged, and glutamic acid was variable in amount
throughout the incubation (Fig. 5).
The Chesapeake Bay incubation showed similar
trends in amino acid distribution to those seen in the
marsh incubation (Fig. 5—middle panel). At 12 h the
bulk of the amino acids was made up by the charged,
polar amino acids (64%), followed by nonpolar amino
acids (33%) and uncharged, polar amino acids (3%). At
the end of the experiment (24 h) the amino acid distribution shifted to 66%, 30%, and 4%, for nonpolar
amino acids, charged, polar amino acids, and uncharged
polar amino acids, respectively. Again there was a small
increase in nonpolar amino acids, a decrease in amino
acids with polar, charged side chains. For the nonpolar
amino acids, glycine and alanine increased significantly
during the incubation, while phenylalanine, leucine,
valine, proline, and isoleucine remained unchanged
(Table 1). All amino acids with polar charged side
chains decreased through the incubation (Fig. 5).
By contrast with freshwater or anoxic waters, amino
acid composition in the lower Delaware Bay incubation
followed a markedly different pattern (Fig. 5—lower
Table 1
Major amino acid composition (as mole percent) of total dissolved protein (5 kDa–0.45 um) fractions collected and concentrated at various time
after addition of BSA to Marsh, Chesapeake Bay, and Lower Delaware Bay waters
Marsh
Chesapeake Bay
Lower Delaware Bay
Collection time (h)
12
24
36
48
12
24
36
48
12
24
36
48
THR
PRO
GLY
ALA
VAL
LEU
ILE
PHE
ASP + ASN
GLU + GLN
LYS
1.51
2.15
3.46
9.68
4.57
2.81
2.51
4.71
4.15
64.45
0.00
8.37
3.69
22.75
10.78
5.76
8.01
1.81
4.94
28.43
4.23
1.23
5.37
7.45
2.28
2.20
3.26
1.01
3.60
6.82
26.33
24.11
17.58
5.18
3.53
32.04
20.28
4.03
5.46
1.35
4.00
17.95
4.69
1.47
3.14
6.29
3.08
3.82
6.20
9.86
1.90
2.06
45.21
10.82
7.63
5.81
4.43
7.15
18.89
3.03
14.25
1.20
8.72
27.99
6.43
2.10
0.03
4.78
11.76
11.26
1.64
4.77
3.20
2.41
16.88
32.77
10.49
3.92
17.94
12.10
13.93
5.55
10.01
3.31
2.80
24.01
4.64
1.81
1.91
3.75
4.14
24.02
6.14
13.28
1.78
9.49
23.88
10.73
0.87
2.09
4.40
7.75
10.89
5.56
10.70
1.51
7.92
34.64
11.94
2.59
3.96
3.46
17.67
16.73
2.85
3.10
1.38
6.00
29.67
8.20
6.98
3.78
3.58
29.50
12.17
5.55
9.63
2.56
6.91
17.48
6.69
2.15
Trp is lost during acid hydrolysis, Met and His were highly variable and excluded.
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7
100
Uncharged, Polar
Non polar
Charged, Polar
Mole Percent
80
60
40
20
0
BSA
12
24
36
48
BSA
12
24
36
48
Abiotic
80
60
40
20
0
Abiotic
100
Mole Percent
80
60
40
20
0
BSA
12
24
36
48
Abiotic
Fig. 5. The amino acid distribution of dissolved protein (defined as N5 kDa) present in Marsh (top panel), Chesapeake Bay (middle panel) and lower
Delaware Bay (bottom panel) waters at various time points over the 48-h incubation time. Amino acids are grouped into major functional groups to
illustrate the changing character of the total protein pool.
panel). The major amino acids after 12 h were the
nonpolar amino acids (63%), followed by the amino
acids with polar charged side chains (35%), and uncharged, polar amino acids (2%). This composition
showed only minor shifts over the incubation period
with a distribution of 70%, 26%, and 4%, respectively.
Among the amino acids with nonpolar side chains,
glycine showed a significant increase while leucine
and alanine showed a significant decrease in the incubation, and valine, isoleucine, proline, and phenylala-
nine remained largely unchanged. Glycine and alanine
were relatively higher by 48 h than BSA with leucine
reduced. For the polar, charged amino acids, aspartic
acid showed a significant decrease over time, while
lysine increased over the incubation and unlike the
other incubations, glutamic acid remained relatively
unchanged. Although lysine appeared to increase over
the incubation period, it remained lower than the BSA
standard at all time points. The overall amino acid
distribution of total hydrolysable amino acids within
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the 5 kDa to 0.45 Am range shows a decrease in
charged, polar amino acids.
3.3.2. Amino acid composition of modified products
Identified products of protein degradation seen in
SEC were collected and analyzed individually. For the
marsh incubations, size-exclusion peaks were collected
based on molecular weight fractions N BSA (100–1000
kDa), b BSA (2–52 kDa), and compared to BSA in
major amino acid composition (Table 2). To compare
changes in overall character, the amino acids were
again grouped by side chain functionality (Fig. 6). In
the marsh incubation at 12 h (Table 2), the dominant
group of THAA in the NBSA fraction were amino acids
with the charged, polar side chains (51%), followed by
nonpolar amino acids (43%), and then the uncharged,
polar amino acids (6%). This distribution is very similar
to that seen for BSA (51%, 42%, and 7%, charged
polar, nonpolar, uncharged polar, respectively). By contrast, modified products with molecular weights less
than BSA at 12 and 24 h were very different from
BSA, with amino acid distribution of 16%, 78%, and
6%, respectively at 24 h. Based on amino acid composition shifts, it can be seen that as the incubation
progresses the individual amino acid composition
more closely resembled BSA.
In the Chesapeake Bay and lower Delaware Bay
incubations, adequate material was available for individual products of BSA hydrolysis at nominal masses at
24 and 48 h to be collected (Table 2). At 24 h the
nonpolar amino acids were dominant (67%), followed
by the charged, polar groups (30%), and then the
uncharged polar amino acids (3%). At 48 h this distribution shifted to 68%, 10%, and 22%, respectively. The
nonpolar amino acid group remained relatively unchanged, while the polar charged group decreased,
and the uncharged polar group increased over time.
The low molecular weight fraction at 48 h was unique
in the increase of uncharged, polar amino acids. This
was the only occurrence of such an increase for this
amino acid group (the rest were below 10%). By the
end of the incubation period, however, the amino acid
composition more closely resembled that of intact BSA
(Fig. 6). In the lower Delaware Bay incubation (Table
2, Fig. 6), the NBSA molecular weight fraction collected at 12 h was similar to that seen in the marsh
incubation. The composition was similar to BSA with
charged polar amino acids (49%) as the dominant
group, followed by nonpolar amino acids (48%), and
uncharged, polar amino acids (3%). The b BSA fraction
was also similar to the marsh incubation, with a distribution of 33%, 64%, and 3% for charged polar, nonpolar and uncharged polar, respectively. In this
incubation, the 66 kDa peak corresponding to BSA
was collected at 24, 36, and 48 h. The distribution of
amino acids in this peak was different than expected for
BSA, with nonpolar amino acids dominating (75%),
followed by uncharged, polar amino acids (17%), and
then charged polar amino acids (8%). Individual amino
acids of the collected peaks did not show a shift over
time towards intact BSA composition as seen in the
other two incubations, but instead showed an overall
Table 2
Major amino acid composition of proteinaceous material collected from size exclusion chromatography of natural water incubation samples at
various time points
Chesapeake Bay
24 h
24 h
Lower Delaware Bay
24 h
48 h
24 h
24 h
Marsh
24 h
36 h
12 h
12 h
12 h
12 h
24 h
45 kDa 29 kDa 17 kDa 12 kDa 165 kDa N66 kDa 13 kDa 66 kDa N100 kDa 66 kDa 44 kDa 5–52 kDa 64 kDa
THR
2.18
PRO
5.59
GLY
26.85
ALA
17.35
VAL
27.71
LEU
4.59
ILE
3.39
PHE
2.49
ASP + ASN
3.02
GLU + GLN 4.17
LYS
2.66
1.35
3.68
21.20
13.84
4.37
4.92
1.72
2.94
18.98
19.29
7.71
3.66
3.14
18.88
19.53
5.51
11.33
4.26
5.22
12.64
12.11
3.73
21.63
4.21
29.37
6.24
14.56
4.70
4.35
1.87
2.09
4.85
6.14
3.04
2.93
16.19
10.16
6.34
5.84
3.25
2.94
13.32
24.02
11.97
2.92
2.67
24.16
13.52
4.88
5.17
2.18
2.12
11.91
19.73
10.75
3.39
6.90
26.71
15.23
4.13
5.03
3.25
2.28
13.67
16.13
3.29
21.63
4.21
29.37
6.24
14.56
4.70
4.35
1.87
2.09
4.85
6.14
1.15
4.83
32.22
4.80
4.59
6.51
1.21
4.50
14.67
20.65
4.87
4.11
3.59
18.25
12.63
6.43
4.82
1.83
6.05
8.93
20.37
13.00
3.44
5.50
18.73
19.48
8.48
4.60
1.70
2.21
11.34
16.30
8.22
6.17
2.08
33.53
20.69
21.46
1.97
1.98
1.37
5.37
2.47
2.89
15.17
18.06
6.79
3.84
16.52
9.69
5.31
4.98
1.18
6.50
11.96
For marsh incubations, peaks within the size ranges of LMW 5–52 kDa, 52–100 kDa, and N100–1000 kDa were combined.
Chesapeake Bay and Lower Delaware Bay samples also contained individual peaks with nominal masses shown. The marsh incubation contained a
combination of 12- and 24-h time points.
Trp is lost during acid hydrolysis; Met and His were highly variable and have been excluded.
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
100
Mole Percent
Uncharged Polar
80
Marsh
Non polar
Charged Polar
60
40
20
0
BSA
100-1000
5-52
52-100
Abiotic
Modified Product Molecular weight (kDa)
Mole Percent
100
Chesapeake Bay
80
60
40
20
0
BSA
45
29
17
Abiotic
Modified product molecular weight (kDa)
100
Mole Percent
Delaware Bay
80
60
40
20
0
BSA
13
>66
48
Abiotic
Modified product molecular weight (kDa)
Fig. 6. The amino acid composition (mol%) of major protein hydrolysis products observed during BSA degradation in marsh and anoxic
Chesapeake Bay waters. After 12 h, modified products were isolated
by size exclusion chromatography, followed by amino acid analysis.
Molecular weight of products that differed were calibrated using
retention times of known proteins. The protein standard and a concentrate from the abiotic incubation at 92 h are included for comparison. See Fig. 3 for illustration of the range of products observed.
further increase in the charged, polar amino acids, and a
further decrease in nonpolar amino acids.
4. Discussion
Size-exclusion chromatography and mass spectrometry of the Patuxent River incubation confirmed that
modified products of protein appear rapidly in the
presence of natural microbial communities. The molecular weight changes observed are consistent with pre-
9
vious reports (Nunn et al., 2003; Pantoja and Lee,
1999), where it was observed that 2–3 amino acids
appear to be initially hydrolyzed from a protein during
microbial degradation, and the modified product then
released into the environment for further attack. In these
studies it was unclear where hydrolytic attack occurred,
but it has been suggested that hydrolysis is initiated
from the terminal ends of the protein (Nunn et al.,
2003). An examination of the primary structure of
BSA (Hirayama, 1990), shows that nonpolar amino
acids are situated on the terminal ends of the protein.
If the termini of a protein are sites for initial hydrolysis,
then we would expect nonpolar amino acids would be
preferentially lost. Rather, the present results of amino
acid analysis suggest the initial loss of charged polar
amino acids. Although the natural conformation of
BSA in solution is not firmly established, its high
solubility suggests hydrophilic, charged polar amino
acids are likely to be located on the exposed face
with more hydrophobic amino acids enclosed. That
being the case, during hydrolysis hydrophilic amino
acids would be removed first, with modified products
tending to have a more hydrophobic character than the
original protein. Previous work by Nguyen and Harvey
(2001) has shown that proteins which have survived
early diagenesis have a more hydrophobic character,
allowing aggregation and the potential for preservation.
This is consistent with our findings of rapid aggregation
of proteinaceous material in all incubations examined
soon after initial hydrolysis.
The present results differ from previous studies with
longer experimental periods and less frequent sampling,
which generally found little selective removal of amino
acids during protein decay (Henrichs and Farrington,
1987; Nguyen and Harvey, 1997; Rosenfeld, 1979;
Wakeham et al., 1984). The preservation of glycine
and serine seen in the present results has been previously observed, but has been attributed to the refractory
nature of diatom cell walls (Hecky et al., 1973; Lee and
Cronin, 1984; Siezen and Mague, 1978). Carlson et al.
(1985) have also suggested that binding of certain
amino acids to macromolecular organic matter may
also permit glycine enrichment. In the present incubations, a clear shift was apparent at the outset of incubations as polar amino acids were preferentially used, yet
as the incubation progressed the overall composition
shifted to more closely resemble the amino acid distribution found in the added intact protein. This suggests
that while there can be selectivity in the earliest stages
of hydrolysis, such selectivity is of short duration as the
remaining modified proteins continue on the path of
mineralization. In the case of BSA, the altered amino
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
acid composition over short incubation times were
transient, with longer incubation times and more degraded products, reflecting an amino acid composition
similar to that of the intact protein. Given that the total
amino acid composition of many proteins is similar, the
analysis of particles or dissolved materials which contain both intact and modified products might be
expected to show only subtle differences in amino
acid composition.
Many studies have examined kinetic rates of bacterial utilization of proteins and amino acids and consequently, protein degradation (Hollibaugh and Azam,
1983; Keil and Kirchman, 1991b, 1993; Samuelsson
and Kirchman, 1990; Kirchman and Hodson, 1984).
Even so, the number of individual proteins identified
in aquatic environments is extremely limited (Tanoue,
1995; Tanoue et al., 1996; Saijo and Tanoue, 2004) and
the enzymatic mechanism(s) for the utilization of protein in these environments are not well understood. The
results here and many others (e.g. Harvey et al., 1995)
support the rapid bacterial degradation of proteins, with
BSA in the present work reduced by over half within
the first 6 h and the majority removed by 12 h in all
experimental replicates. Yet even over these short time
scales, new proteinaceous intermediates were observed,
having both greater and lower masses than the protein
added. A number of these products appear to be related
to BSA by sequential loss of amino acids (Fig. 4). Our
results also suggest that as protein is hydrolyzed, intermediates released back into the environment are available for further degradation. These results are consistent
with recent work in sediments suggesting that not all
intermediate products formed from protein degradation
are immediately taken up, but instead some were released back to the water (Nunn et al., 2003). Vetter et al.
(1998) suggested a similar model in which proteins in
sediments are partially hydrolyzed and the remaining
product released.
The enzymatic mechanisms of protein degradation
in natural waters are important in determining rates
and patterns of dissolved protein degradation. Several
models exist that describe the bacterial degradation of
protein, including those suggesting that extracellular
enzymes are released to hydrolyze proteins (Mayer et
al., 1995). The small molecular weight changes observed in the protein incubations here are consistent
with extracellular hydrolysis, indicative of the action
of an exoprotease, which hydrolyze amino acids from
the termini of proteins (Vetter et al., 1998). The results
here show that while BSA was degraded rapidly
through the experimental incubation, some high molecular weight products were also formed. The pres-
ence of aggregated high molecular weight products
have been observed during protein degradation
(Nguyen and Harvey, 2001) and hydrophobic interactions and/or hydrogen bonding have been suggested as
possible mechanisms for its preservation. Additional
modifications may occur under abiotic conditions
(Keil and Kirchman, 1993), but are observed on longer temporal scales.
Comparisons of the three estuarine sites suggest that
environmental differences and bacterial community
structure had little effect on the production of protein
degradation products. Although there were variations
among the intermediate products produced, all incubations showed a rapid decrease in added protein, indicating that differences in the bacterial community had
little effect on relative degradation rates. In parallel
work, Harvey et al. (in press) has observed that the
addition of BSA to natural water incubations at related
sites showed no consistent shifts in bacterial communities based on 16S RNA phylogenetic analysis (FISH).
(Cottrell and Kirchman, 2000) have shown similar
results in the examination of DOM utilization and
bacterial community shifts in estuarine and coastal
environments in that utilization did not correlate with
shifts in the abundance of specific bacterial groups.
This may not be the same for other classes of organic
carbon. For example, Arnosti (2004) has discussed the
case for polysaccharides; detailing experimental evidence that initial enzymatic hydrolysis is not always
the slow step in subsequent degradation. Enzyme production (or its limitation) and varied extracellular enzyme distribution were also suggested as factors which
might regulate organic matter degradation. These varied
responses suggest that while there appears sufficient
breadth among the microbial communities in these
diverse environments to utilize added proteins and
their products, this might not be the case for other
fractions of organic matter.
Although overall protein degradation was rapid, the
pattern of high molecular weight products seen was not
uniform among the three environments (Fig. 3). If
dissolved protein complexes with organic matter creating more refractory, high molecular weight material, we
would speculate that the marsh incubation would have
the highest aggregation rates given its high DOM content, followed by the Chesapeake Bay and lower Delaware Bay with the lowest organic content. The marsh
did show the greatest relative abundance of high molecular weight proteinaceous material; by 48 h the
losses were similar to the other sites. The organic matter
content of the environments may influence the degree
of overall aggregation into high molecular weight pro-
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
teinaceous material, however further studies are required to investigate what effect this may have on the
potential for preservation.
BSA is a unique protein in that it has a somewhat different amino acid composition than many
aquatic proteins (Nguyen and Harvey, 1994b), and
together with commercial availability of the highly
purified protein allowed small changes in molecular
weight modification to be followed in the present
study. The amino acid composition shifts seen in
BSA were most similar in the marsh and the Chesapeake Bay incubations. Nevertheless, in all three
sites modified products more closely resembled the
amino acid composition of BSA as the incubation
progressed, despite initial shifts in functional groups.
This indicates a selective attack on the protein with
the initial removal of charged polar amino acids and
retention of nonpolar amino acids, specifically glycine and alanine. Although the natural conformation
of BSA is not known, we can speculate that these
charged polar amino acids occupy the outer shell of
BSA when it is in its natural hydrated conformation.
After these amino acids are removed and the interior of the protein is exposed, remaining amino
acids are utilized. Support for this hypothesis can
be seen in the products collected over time suggesting that charged polar amino acids are depleted and
there is a corresponding increase in glycine, indicating preservation. The higher molecular weight intermediates (N 100 kDa) observed were very similar in
amino acid composition to BSA. This material may
contain significant amounts of native BSA or that
with minor alterations caused by small changes in
the chemical properties of BSA through degradation.
It is interesting to note that different phytoplankton
have all been found to have very similar amino acid
compositions (Cowie and Hedges, 1992; Nguyen and
Harvey, 1994b). Dissolved combined amino acids in
various waters all had very similar amino acid compositions, including the Delaware estuary (Keil and
Kirchman, 1993), particulate matter from surface oceanic and coastal waters (Siezen and Mague, 1978),
Peru coastal upwelling waters (Wakeham et al.,
1984), and particulate organic matter in Arctic and
Antarctic waters (Hubberten et al., 1995). Although
similar amino acid compositions exist in most environments due to similarity in amino acid protein
composition, attack by bacteria may first require
selective attack of external amino acids, namely the
charged, polar amino acids. Once the interior of the
proteins is exposed, all amino acids are used at equal
rates.
11
5. A conceptual model of protein structure during
degradation
While information on specific protein modification
is abundant in the biomedical literature (e.g. signaling
proteins or activation) the degradation process is less
specific. As a soluble, globular protein, it requires that
the hydrophilic (polar, charged) amino acids of BSA are
largely located on the exterior. The hydrophobic, nonpolar amino acids are directed towards the interior and
the uncharged, polar amino acids throughout the structure. As the protein is utilized, smaller, modified products are formed along with aggregated higher
molecular weight material. From the amino acid analysis, it is apparent that as the protein is utilized, there is
a loss of charged, polar amino acids. As these amino
acids are removed, the tertiary structure of the protein is
compromised and the protein might unfold, exposing
the interior hydrophobic amino acids. Once the protein
unfolds, there is no selective attack on amino acids with
utilization limited largely by the protein conformation.
There does not appear to be selective attack on uncharged, polar amino acids, supporting the suggestion
that the differences in the utilization of these amino
acids are selective due to amino acid availability within
a variable protein conformation. This suggests that one
explanation for the increased aggregation could be the
exposure of the interior nonpolar amino acids. Aggregates would form through hydrophobic interactions
among nonpolar amino acids again surrounded by
charged polar amino acids.
6. Conclusions
Although proteins are degraded rapidly in natural
waters, intermediates of varied molecular weight can
be observed over short time periods. Amino acid analysis of a suite of these intermediate products shows that
charged, polar amino acids appear preferentially lost
from the model protein BSA with an increased fraction
of nonpolar amino acids present in such modified products. Over time and with additional degradation, the
amino acid composition returned to that more closely
resembling the amino acid composition of the intact
protein. Initial products also suggest that that the protein
may not be hydrolyzed from the end terminus, but
instead that the charged, polar amino acids present at
multiple sites on the outer surface of the protein in its
natural conformation are removed. As these amino acids
are removed from the protein, the interior amino acids
are exposed and utilized, resulting in little selective
preservation. Further examination using a range of tech-
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L.C. Roth, H.R. Harvey / Marine Chemistry xx (2006) xxx–xxx
niques (e.g. Ostrom et al., 2000) and sequencing of
specific products is needed to identify amino acid locations in natural conformations of proteins to verify the
initial sites of hydrolysis in the environment.
Acknowledgements
This work was supported by the Chemical Oceanography program of the NSF. Rachael Rearick is
thanked for technical assistance with protein incubations and Angela Squier for advice and manuscript
comments. Constructive comments by two anonymous
reviewers substantially improved the final manuscript.
Contribution No. 3926, University of Maryland Center
for Environmental Science.
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