surface reactions on mineral particles controlling the hydrolysis...
TRANSCRIPT
i
SURFACE REACTIONS ON MINERAL
PARTICLES CONTROLLING THE
HYDROLYSIS OF GLUCOSE PHOSPHATES
RICKARD OLSSON
DEPARTMENT OF CHEMISTRY
UMEÅ UNIVERSITY
SWEDEN
2011
ii
COPYRIGHT©2011
ISBN: 978-91-7459-270-2
PRINTED BY KBC-TRYCKERIET
UMEÅ UNIVERSITY
UMEÅ, SWEDEN 2011
iii
Abstract
Phosphorus (P) is an essential nutrient. A significant amount of soil P may be in the form of
organophosphates. Due to the size of these compounds, hydrolysis is often required before P
can be assimilated by organisms. Hydrolysis may be mediated by mineral surfaces, or
catalyzed by extra cellular enzymes. Since both organophosphates and enzymes have a strong
affinity for environmental particles, a study of the hydrolysis of organophosphates must focus
on reactions at the water/particle interface. This thesis is a summary of four papers, discussing
the adsorption, desorption, and abiotic and enzymatic hydrolysis of glucose-1-phosphate
(G1P) and glucose-6-phosphate (G6P) in aqueous goethite suspensions. A new technique for
simultaneous infrared and potentiometric titrations (SIPT) allowed in-situ measurements of
the interfacial reactions. It was found that glucose phosphates form pH-dependent inner
sphere complexes on goethite, which coordinate in a monodentate fashion, and are stabilized
by hydrogen bonding. Desorption involves a change in speciation of the surface complexes,
illustrating the difficulty in determining desorption rates for individual complexes. The
surface mediated hydrolysis is primarily base catalyzed for G1P, and acid catalyzed for G6P.
The difference is partly due to electronic factors, and partly to differences in glucose
group/goethite interactions. Considerably more extensive is the hydrolysis catalyzed by an
acid phosphatase (AcPase). The rate of the enzymatic hydrolysis are strongly dependent on
the glucose phosphate surface coverage, showing that surface properties affect the adsorption
mode of enzymes, and thus their catalytic activity. In solution, AcPase showed a greater
specificity towards G6P, but this specificity was partly lost after adsorption onto goethite.
iv
Surface Reactions on Mineral Particles Controlling the Hydrolysis of
Glucose Phosphates
This thesis contains a summary and a discussion of the following papers.
I. Adsorption, Desorption, and Surface-Promoted Hydrolysis of Glucose-1-Phosphate in
Aqueous Goethite (α-FeOOH) Suspensions
Rickard Olsson, Reiner Giesler, John S. Loring, and Per Persson. Langmuir, 2010, 26 (24),
18760-18770. Reprinted with permission, copyright 2010 American Chemical Society.
II. Enzymatic Hydrolysis of Organic Phosphates Adsorbed on Mineral Surfaces
Rickard Olsson, Reiner Giesler, John S. Loring, and Per Persson. Submitted to Environmental
Science & Technology.
III. Abiotic and Enzymatic Hydrolysis of Glucose-6-phosphate on Goethite Particles
Rickard Olsson, Reiner Giesler, Malin Lindegren, and Per Persson. Manuscript.
IV. Adsorption Mechanisms of Glucose in Aqueous Goethite Suspensions
Rickard Olsson, Reiner Giesler, and Per Persson. Journal of Colloid and Interface Science,
353, 2011, 263-268. Reprinted with permission from Elsevier.
v
Contents
Introduction ......................................................................................................................................... 1
Aim ...................................................................................................................................................... 3
PART I Adsorption, Desorption, and Hydrolysis of Glucose Phosphates in Goethite Suspensions .... 3
1. Experimental Procedures and Techniques....................................................................................... 3
1.1 Batch Experiments .................................................................................................................... 3
1.2 Simultaneous Infrared and Potentiometric Titration (SIPT) ..................................................... 3
1.3 ATR FTIR Spectroscopy ........................................................................................................... 5
1.4 Two-Dimensional (2D) Infrared Correlation Spectroscopy ...................................................... 6
3. Infrared Spectroscopic Characterization of G1P and G6P Surface Complexes .............................. 9
3.1 Assignment of Peaks in Infrared Spectra of Aqueous G1P and G6P ........................................ 9
3.2 The Structure of G1P and G6P Surface Complexes ................................................................ 10
5. Surface Promoted Hydrolysis of G1P and G6P Adsorbed onto Goethite ..................................... 19
6. Enzymatic Hydrolysis of G1P and G6P Adsorbed onto Goethite ................................................. 22
PART II Adsorption mechanisms of glucose in aqueous goethite suspensions .................................... 29
1. Introduction ................................................................................................................................... 29
2. Adsorption of Glucose on Goethite ............................................................................................... 30
3. FTIR Characterization of Glucose Adsorbed onto Goethite ......................................................... 30
Conclusions ........................................................................................................................................... 34
References ............................................................................................................................................. 35
Acknowledgements ............................................................................................................................... 37
1
Introduction
Phosphorous (P) is essential to life, and is found in DNA, RNA, ATP, and phospholipids. In
the environment, P exists almost exclusively as inorganic or organic phosphates, thus the
biochemistry of P is, to a considerable degree, controlled by the properties of the phosphate
group.[1, 2] Phosphate groups possess an affinity for environmental particles, and especially
strong is the reactivity towards those particles containing Fe, Mn, and Al.[1, 3-5] Therefore,
the study of the biogeochemical cycling of P naturally focuses on reactions at the
particle/water interface.
Organophosphates may constitute a substantial part of the total P in soils.[6, 7] Their
abundance makes them an important potential P source for organisms. In organic phosphates
the phosphate group is bonded to organic molecules via phosphate ester bonds. Due to the
size of organophosphates, hydrolysis of the ester bond is often a necessary step in order to
make organic P available for uptake by plants and microorganisms. Also, since
organophosphates contain elements such as C and N, hydrolysis also releases other elements
necessary for growth. For example, the addition of glucose-6-phosphate causes bacteria to
produce enzyme to aid them in glucose utilization.[8] The hydrolysis of organophosphates is,
therefore, of interest not only in a P bioavailability perspective, but also regarding, for
example, the mobility of the earth’s soil carbon pool, which is an issue currently discussed in
the context of climate change.
Hydrolysis of organophosphates is either abiotic or enzymatic. The abiotic process is
mediated by the surface of metal oxides. Organophosphorus pesticides have been found to
hydrolyze on goethite (α-FeOOH), TiO2, Al2O3, and Al(OH)3, in some cases forming
persistent and hazardous compounds.[9, 10] Iron and manganese oxides have been found to
hydrolyze para-nitrophenyl phosphate, which is commonly used as a model compound in
studies of organophosphates.[11, 12]
Enzymes that catalyze the hydrolysis of organophosphates include acid phosphatases, alkaline
phosphatases, and phytases.[13-15] A further reason to focus on interfacial reactions in the
2
study of the hydrolysis of organophosphates is that enzymes, like phosphates, have an affinity
for environmental particles.[14, 15] Adsorption on soil particle surfaces may reduce
enzymatic activity, but may also protect the enzymes, thus making them more persistent.[16,
17]
One group of organophosphates is the phosphorylated sugars. Two examples of these,
examined in the present study, are the structurally similar glucose-1-phosphate (G1P) and
glucose-6-phosphate (G6P), shown in figure 1. G1P and G6P are biochemically important
molecules involved in cell metabolism, and studies indicate that sugar monoesters like these
may occur at significant concentrations in soils.[18, 19] The choice of G1P and G6P as model
compounds provides an opportunity to systematically study how minor structural differences
affect adsorption and desorption, as well as the abiotic and enzymatic hydrolysis.
Figure 1. Lewis structures of (a) glucose-1-phosphate and (b) glucose-6-phosphate.
In Part I of this work the interactions of G1P or G6P with the goethite surface are examined.
The mineral goethite (α-FeOOH) is an iron oxohydroxide that due to its thermodynamic
stability is found in all types of soils throughout the world.[20] The enzymatic hydrolysis of
G1P or G6P adsorbed onto goethite is also addressed in Part I, using an acid phosphatase
(AcPase) from potato. Acid phosphatases are a rather non-specific group of enzymes and can
catalyze a number of phosphate esters.[21, 22] The pH optimum of the AcPase used is 5.0-
5.3.[21]
The hydrolytic products of G1P or G6P are phosphate and glucose, and while the adsorption
of phosphate onto goethite has been the subject of numerous studies, less has been written
1 6
a) b)
1 6
3
about the interaction between glucose and the goethite surface. The adsorption of glucose
onto goethite is examined in Part II of this study.
Aim
The primary aim of this study is to, on macro- and molecular levels, examine the enzymatic
and abiotic hydrolysis of glucose-1-phosphate and glucose-6-phosphate, adsorbed onto
goethite. In order to do so, it was necessary to also study the adsorption and desorption
reactions of the ligands in goethite suspensions. The interactions of glucose, i.e. one of the
hydrolytic products, with the goethite surface were also studied.
PART I Adsorption, Desorption, and Hydrolysis of Glucose Phosphates in
Goethite Suspensions
1. Experimental Procedures and Techniques
1.1 Batch Experiments
The adsorption experiments were carried out in batch. The pH of a goethite suspension was
adjusted to a target pH in the range 3 to 10, then G1P or G6P was added, and pH adjusted
again. The final goethite concentration was 10 g/L. The samples were continuously purged
with N2(g) during sample preparation in order to avoid CO2 and related carbonate
contamination. After 1 to 48 hours on an end-over-end rotator, the samples were centrifuged
and filtrated. The supernatant was analyzed for G1P, G6P, orthophosphate, or glucose, using
ion chromatography. Infrared spectra of supernatant and paste were collected, and the latter
was subtracted from the former to eliminate the contribution from water and aqueous species.
1.2 Simultaneous Infrared and Potentiometric Titration (SIPT)
Infrared spectra were collected during the enzymatic and desorption experiments using a
setup for simultaneous infrared and potentiometric titrations (SIPT). The setup is shown
schematically in figure 2, and has been described by Loring et al.[24] A goethite suspension
was pumped peristaltically in a closed loop between a titration vessel and a flow-through
ATR cell. In the ATR cell the goethite suspension is flowed over a thin goethite film (aka, the
overlayer) which is deposited on a ZnSe crystal. G1P or G6P was added to the titration vessel,
and was allowed to adsorb for ca. 2.5 hours before AcPase was added. Infrared spectra were
4
continuously collected, first to monitor the adsorption process, and then to follow the
hydrolytic reaction.
Figure 2. Schematic view of the setup for simultaneous infrared and potentiometric titrations (SIPT).
G1P and G6P were added at pH 9.4, and then the pH was slowly brought down to the targeted
pH at 4.5, 5.0, or 5.5. The aim of this procedure is to achieve a homogenous distribution of
the ligand on the goethite particles in suspension as well as on the overlayer. The pH was kept
constant with an automated and computer controlled burette system, and thereby the use of
buffers, which otherwise may have an effect on the interfacial processes, could be
avoided.[25] With a homogenous distribution, reactions on the overlayer are representative of
reactions in the suspension. The purpose of the overlayer is to collect goethite close to the
ZnSe/suspension interface, where the IR radiation is most intense. This means that the bulk of
the signal in the infrared spectra is from the ligand adsorbed on the goethite in the overlayer,
and the signal from the ligand adsorbed on the goethite in the suspension is negligible.
In order to quantitatively follow the hydrolysis reaction, samples were collected from the
titration vessel, and were immediately analyzed for glucose using ion chromatography.
The SIPT setup was also used in desorption experiments. After addition of G1P or G6P,
followed by equilibration, the peristaltic pump was stopped, and the goethite suspension in
the titration vessel was replaced by a ligand-free goethite suspension, then the pumping was
resumed. The first 10 mL of the ligand-free suspension pumped into the flow-through cell
was discarded; it was used only to flush the ligand-containing suspension out of the cell. After
the pump was started, spectra were continuously collected as the ligand desorbed from the
overlayer and adsorbed onto the goethite particles in suspension.
FTIR
w/ATR Cell
Dosimeter with
Potentiostat
Computer
Propeller Stirrer
Titration Vessel
Peristaltic Pump
Combination pH Electrode
5
1.3 ATR FTIR Spectroscopy
In spectroscopy the interaction of electromagnetic radiation with matter is studied. The
frequencies of infrared (IR) radiation (400-7000 cm-1
) overlap with the frequencies of
molecular vibrations. When a molecule is irradiated with infrared light at a frequency
corresponding to that of its vibration, energy is absorbed, provided that the vibration changes
the dipole moment of the molecule. A molecule consisting of more than two atoms has more
than one vibrational mode, and may adsorb infrared light at different frequencies.
Using an harmonic oscillator as a model for the vibration of a diatomic molecule containing
the atoms A and B, we see that the frequency of the molecular vibration, usually expressed as
the wavenumber (ω), depends on the mass of atoms involved, and the force constant of the
bonds (k):
ω = 1/2π (k/µ)1/2
where µ is the reduced mass of the atoms, i.e. µ = mAmB/mA + mB, where m is the mass of the
atom. The wavenumber (ω) is the reciprocal of the wavelength (λ): ω = 1/λ. The wavelength
in turn is related to the frequency (ν) according to νλ = c, where c is the speed of light.
A certain combination of atoms will vibrate with a certain frequency, and absorb infrared
light at the corresponding wavenumber. The vibrational frequency of a group of atoms may
be affected by interactions with other molecules, since the interactions may change the
strength of the atomic bonds, which affects the force constants, or the symmetry of the
molecule and therefore the dipole moment. In the present work infrared spectra are interpreted
in order to gain molecular-level knowledge about adsorption, desorption, and hydrolysis
reactions at the goethite surface.
FT in FTIR stands for Fourier Transform, which is the mathematical procedure that allows the
whole infrared spectrum to be recorded at once. Advantages of this are speed, improved
signal-to-noise ratio, and superior frequency accuracy.[23]
Attenuated Total Reflectance (ATR) is a widely used technique in the field of FTIR
spectroscopy. In ATR sampling, the IR light beam penetrates the sample to a shallow depth.
As a consequence, little or no sample preparation is required, which contrasts with
transmission spectroscopy where the sample needs to be diluted with an IR transparent salt
like KBr.
6
In ATR spectroscopy the IR beam is sent through a crystal referred to as the Internal
Reflection Element (IRE), and undergoes total internal reflection at the interface with the
sample, provided that: 1) the sample has a lower refractive index (n), and 2) the angle of
incidence (θ) exceeds the critical angle.
The relation between the critical angle (θc), and the refractive indices of the sample (n2) and
the crystal (n1) is:
θc = sin-1
(n2/ n1)
If the angle of incidence does not exceed the critical angle, there will be some external
reflection as well. The presence of derivative shaped peaks in the infrared spectrum may be an
indication of this.
When the IR beam undergoes internal reflectance, an evanescent wave projects into the
sample (figure 3). The sample absorbs energy at different wavenumbers and alters the
evanescent wave accordingly.
The depth of penetration (dp) of the IR beam into the sample is (arbitrarily) defined as the
distance required for the amplitude of the evanescent wave to fall to e-1
of its value at the
interface. Values for dp range from about 0.5 to 5 microns, but the definition indicates that the
actual sampling depth can be greater. Sampling depth decreases as the angle of incidence
increases.
Figure 3. Schematic view of attenuated total reflectance (ATR).
1.4 Two-Dimensional (2D) Infrared Correlation Spectroscopy
In 2D infrared correlation spectroscopy, conventional infrared spectra are spread in two
dimensions.[26] The 2D analysis provides information on correlation of peaks, which is
helpful in assignment, as well as reveals the sequential order of changes in peak intensities. In
Reflected radiation
Evanescent wave
Incident radiation
Sample
Crystal
θ
7
addition, the resulting 2D spectrum consists of sharper and better resolved peaks than in the
original spectra, and thus the 2D analysis may supply information that is not apparent in
conventional spectra. It should be noted that the 2D analysis can be applied in any type of
spectroscopy.
The 2D analysis displays changes in spectral intensities brought on by some kind of
perturbation, e.g. a change of pH, temperature, concentration, or pressure. In the present work,
2D analysis is used to detect spectral variations following addition of ligand or enzyme, as
well as changes as a function of pH.
Two components, the synchronous and the asynchronous correlation intensities, make up the
2D correlation spectrum. The synchronous correlations identify coincidental trends in spectral
changes, while the asynchronous correlations reveal spectral changes that are out-of-phase
with each other. The perhaps bewildering terminology was adopted for purely historical
reasons: early on, the 2D correlation analysis was predominantly used to study time-
dependent variations.
The 2D spectrum can be presented as synchronous and asynchronous maps. In the
synchronous contour map, the diagonal peaks are autopeaks. These are always positive and
indicate at which wavenumbers the major spectral changes occur. Off the diagonal, cross
peaks are found, indicating correlated changes. If a cross peak is positive, the infrared peaks
change in the same direction, i.e. display a simultaneous increase or decrease in intensity. A
negative cross peak means that the infrared peaks change in opposite directions, i.e. one peak
increase in intensity as the other decrease.
The asynchronous map display spectral changes that occur out-of-phase in relation to each
other. Furthermore, the sign of the asynchronous peak provide information on the sequential
order of events. According to Noda’s rule[26], if the asynchronous peak is positive then the
infrared peak defined by the x-coordinate undergoes a change before the infrared peak at y. In
the case of a negative asynchronous signal the relationship is reversed. This rule holds as long
as the synchronous signal is positive, and is reversed if the signal is negative.
8
2. Adsorption of G1P and G6P on Goethite
G1P and G6P each have two pKa values, determined to be 1.2 and 6.1, and 1.3 and 6.2,
respectively. They are thus anionic within the studied pH range (3-10) and exist in solution in
either a mono- or divalent state (HL- and L
2-, respectively).
The acidic properties of the phosphate moiety on G1P and G6P give these compounds
adsorption characteristics which are similar to anions, i.e. adsorption onto goethite decreases
with increasing pH (figure 4).[27] The decrease in adsorption is due to competition with
hydroxide ions, and a decrease in surface charge. GP, however, have a considerably lower
affinity for the goethite surface than orthophosphate, as seen by the steep decline of the
adsorption curves at high pH values. At lower pH values more than 98 % of the added ligand
is adsorbed after one hour. Nevertheless, ligand adsorption occurring over a wide pH range,
including above the isoelectric point of goethite (IEPgoethite= 9.4), indicates a strong interaction
with the mineral surface.
The adsorption of G1P and G6P onto goethite is a fast process, approaching equilibrium
within one hour. The quick adsorption indicates that GP forms surface complexes on goethite,
since surface transformation and surface precipitation reactions are typically slower.[28]
Figure 4. Adsorption of G1P (left) and G6P (right) on goethite as a function of pH and time. Samples with
a total concentration of 1.38 µmol/m2 of goethite are denoted (1 h), (6 h), (24 h), and (48 h).
Samples with a total concentration of 0.69 µmol/m2 of goethite are denoted (1 h), (6 h), (24 h), and
(48 h). Left: denotes adsorbed orthophosphate at a total concentration of 1.53 µmol/m2 of goethite,
after an equilibration time of 24 h.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
2 3 4 5 6 7 8 9 10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
2 3 4 5 6 7 8 9 10
pH pH
µm
ol l
iga
nd
ad
s p
er
m2
of
go
eth
ite
µm
ol l
iga
nd
ad
s p
er
m2
of
go
eth
ite
G1P G6P
9
3. Infrared Spectroscopic Characterization of G1P and G6P Surface Complexes
3.1 Assignment of Peaks in Infrared Spectra of Aqueous G1P and G6P
The determination of the structure of G1P and G6P surface complexes begin with the analysis
of the infrared spectra of the aqueous GP species (HL- and L
2-). These are shown in figure 5.
Both the phosphate (the P-O stretching modes) and glucose moieties produce peaks in the
frequency region 800 – 1300 cm-1
(for the assignment of peaks in spectra of aqueous glucose,
see Part II below). However, according to the DFT calculations little coupling occurs between
the two; this may be due to the large mass of the phosphorous atom which acts as a partial
coupling barrier. Therefore, peaks in this region originate primarily from vibrations of either
the phosphate or the glucose group. Apart from peak shifts, the main P-O stretching modes of
the HL- and L
2- species of G1P and G6P, labeled with an asterisk in figure 6, are similar to
those of H2PO4- and HPO4
2-, respectively (Paper I and III).
Figure 5. Infrared spectra of glucose phosphates in aqueous solution. (a) monoprotonated G6P (HL-), (c)
deprotonated G6P (L2-
), (c) monoprotonated G1P (HL-), (d) deprotonated G1P (L
2-). The main P-O
stretching modes are labeled with (*).
wavenumber/cm-1
abso
rban
ce
d
c
b
a
*
*
*
**
*
10
In table 1, the peaks in the G1P spectra are assigned to vibrational modes based on
comparison with the infrared spectra of HPO42-
(aq), H2PO4- (aq), and glucose (aq), as well as
the theoretical spectra of G1P, hydrated by 10 water molecules (Paper I). For brevity, the
assignment of G6P peaks is not shown.
Table 1. Experimental infrared frequencies (in cm-1
) of the non- and mono-protonated forms of glucose-1-
phosphate in aqueous solution together with tentative group assignments.
3.2 The Structure of G1P and G6P Surface Complexes
3.2.1 Infrared Spectra of G1P and G6P Adsorbed onto Goethite
For a thorough discussion on the structure of the G1P surface complexes, which also applies
to G6P, see paper I. The infrared spectra of G1P and G6P adsorbed onto goethite differ
significantly from the spectra of the aqueous species (figure 6 and 7). There are, however,
some similarities. For example, a number of peaks in the adsorbed spectra as well as in the
corresponding second derivatives (not shown) are in close agreement with peaks in the
aqueous spectra, originating in glucose modes (table 1). For that reason, the new peaks
appearing in spectra of the surface complexes are attributed to P-O stretching modes. This
indicates that the P-O bonds of GP are considerably distorted when the molecule adsorbs, via
L2-
HL- Group assignment
1190 Phosphate mode
1145 1147 Glucose mode
1112 1114 Glucose mode
1094 1088 Phosphate mode
1055 1053 Glucose mode
1026 1030 Glucose mode
993 1009 Glucose mode
967 957 Glucose mode
945 919 Phosphate mode
864 871 Phosphate mode
11
its phosphate group, onto the goethite surface. This distortion is a sign of inner sphere
complexation, i.e. a direct interaction between the phosphate group and the goethite
surface.[29, 30] The P-O peaks in the adsorbed spectra are pH dependent and shift gradually
as the pH changes, indicating at least two different surface complexes.
Figure 6. Infrared spectra of G1P adsorbed on goethite at pH (a) 2.99, (b) 4.04, (c) 4.95, (d) 6.16, (e) 7.16,
(f) 8.11, and (g) 9.04. The reaction time was 48 h and the total concentration of G1P was 1.38 µmol/m2 of
goethite. The ordinate scale is arbitrary and has been adjusted for each spectrum to facilitate qualitative
comparisons.
Figure 7. Infrared spectra of G6P adsorbed on goethite at pH (a) 2.78, (b) 3.95, (c) 5.33, (d) 7.10, (e) 7.63,
(f) 8.66, and (g) 9.22. The total concentration of G6P was 1.36 µmol/m2 of goethite. The ordinate scale is
arbitrary and has been adjusted for each spectrum to facilitate qualitative comparisons.
1250 1200 1150 1100 1050 1000 950 900
ab
so
rba
nce
wavenumber/cm-1
a
b
c
d
e
f
g
abso
rban
ce
wavenumber/cm-1
12
3.2.2 Two-Dimensional (2D) Correlation Analysis of Infrared Spectra of G1P Surface
complexes
The following discussion focuses on G1P, however, data indicate that the conclusions also
applies to G6P (see papers I and III). The 2D correlation analysis of the G1P-goethite spectra
indicates that a minimum of two surface complexes exist. Nine auto peaks are visible in the
synchronous 2D contour map, and according to the cross peaks they form two groups (figure
8 and table 2). Mutually positive cross peaks at 965, 1046, 1058, 1078, 1098, and 1129 cm-1
are negatively correlated to the 986, 1015, and 1160 cm-1
peaks, and vice versa. The
correlated peaks at (1015, 1160) and (1046, 1058, 1129) appear in the region of major spectral
change, and are thus likely to originate predominately in P-O stretching modes.
Figure 8 Synchronous (left) and asynchronous (right) contour maps obtained from a 2D correlation
analysis of the infrared spectra presented in figure 6a-f. The abscissa and ordinate scales are given in cm-1
and the white and grey areas denote positive and negative responses, respectively. The arrows high-light
the weak cross peaks associated with the characteristic distorted butterfly pattern.
13
Table 2. Summary of the synchronous 2D correlation spectroscopy results of G1P adsorbed onto goethite.
The auto peaks along the diagonal (from upper right to lower left) and the off-diagonal cross peaks are
given in cm-1
.
From figure 6 it is not obvious that the 2D contour map peaks at 965, 986, 1078, and 1098
cm-1
are shifting. Instead, it is likely that they are the result of intensity variations relative to
the other peaks. These relative variations could be caused by normalization, and/or by
changes in the absorption coefficients. Comparing the peaks with those of aqueous G1P
(table 1), they presumably originate from the glucose moiety. Since the peaks ascribed to
vibrational modes of the glucose moiety do not seem to change when G1P adsorbs to goethite,
either the 1046 or the 1058 peak may also be due to glucose modes. The uncertainty is due to
complexities in the regular spectra, such as overlapping and broad peaks, which in the 2D
analysis result in peaks with slightly different wavenumbers.
The asynchronous contour map suggests that there are more than two surface complexes. In
the region between 1010 and 1060 cm-1
a characteristic pattern, the so called distorted
butterfly, is seen (figure 8). This asynchronous feature, together with a pair of weaker cross
peaks, indicates that the peaks simultaneously shift and vary in width. Such complex changes
suggest one or more additional intermediate complex.
3.2.3 Structural Assignment of G1P and G6P Surface Complexes
As seen above, G1P and G6P form more than two inner sphere surface complexes when
adsorbing to goethite in the pH range 3 to 10.
965 986 1015 1046 1058 1078 1098 1129 1160
965 + - - + + + + + -
986 - + + - - - - - +
1015 - + + - - - - - +
1046 + - - + + + + + -
1058 + - - + + + + + -
1078 + - - + + + + + -
1098 + - - + + + + + -
1129 + - - + + + + + -
1160 - + + - - - - - +
14
In the spectra in figures 6 and 7, we see that the highest wavenumber P-O stretching peak is
shifted from ca. 1130 to ca. 1150 cm-1
for G1P, and ca. 1114 to ca. 1134 cm-1
for G6P, when
pH decreases. Shifts like these are observed as phosphate or arsenate, or metal complexes of
these ions, are gradually protonated.[24, 29] The shifts are explained by the fact that the
highest frequency P(As)-O peak originates from vibrations involving the strongest P(As)-O
bonds. These are the P(As)-O groups that are not protonated or bonded to metal ions. When
the other oxygen atoms are protonated, their bond strength increases, resulting in a shift of the
highest wavenumber P(As)-O stretching peak to higher frequencies. If then also the last “free”
P(As)-O group would be protonated, the peak would undergo a significant red-shift (i.e. shift
to lower wavenumber). Similar to this case, a bidentate glucose phosphate surface complex
would have only one P-O group available for protonation, and the lack of red-shift in the
spectra tells us that a bridging bidentate model is unlikely.
The gradual shift from ca. 1130 to ca. 1150 cm-1
(G1P), or from 1114 to ca. 1134 cm-1
(G6P)
observed when pH decreases thus suggests a gradual protonation of one of the free P-O
groups. This is consistent with a model in which the G1P surface complex interacts via
hydrogen bonding to a neighboring site. The importance of hydrogen bonding in surface
complexes formed by phosphate and arsenate as they adsorb to goethite has been shown in
recent studies.[24, 31, 32] Although it cannot be ruled out, a direct protonation of the glucose
phosphate surface complexes seems unlikely since this should shift the highest wavenumber
peak above 1196 cm-1
(G1P) or 1184 cm-1
(G6P), which are the positions of the highest
wavenumber P-O stretching peaks of the aqueous HL- species.
Proposed structures of the G1P surface complexes are shown in figure 9: Monodentately
coordinated G1P interacts via hydrogen bonding to a neighboring site. At high pH values,
G1P acts as a hydrogen bond acceptor. With decreasing pH, the proton involved in the
hydrogen bond is gradually shifted towards the oxygen of G1P. As a result, G1P acts as a
donor at low pH values. Corresponding interactions are found in the G6P surface complexes.
15
Figure 9. Proposed structures of G1P surface complexes on goethite. The dotted red lines denote hydrogen
bonding.
4. Desorption of G1P and G6P from Goethite
Desorption of G1P and G6P from goethite is strongly pH dependent (figure 10). At pH 5, a
dependency on the total concentration is also observed, with initially faster desorption at the
higher concentration.
Figure 10. Normalized integrated peak areas of G1P and G6P adsorbed on goethite, as a function of time.
() total concentration of 0.69 µmol/m2 of goethite, and () total concentration of 1.36 µmol/m
2 of
goethite, both at pH 5.0. () total concentration of 1.36 µmol/m2 at pH 8.5.
The data for G1P and G6P are very similar (papers I and III). The following discussion
focuses on G1P, but applies also to G6P. Infrared spectra show that the initial surface
Fe
OH
Fe
H
Fe
OH
FeFe
OH
Fe
H
Increasing pH
SC I SC II SC III
Pe
ak a
rea
no
rmal
ize
d to
are
a at
t=
0 h
Pe
ak a
rea
no
rmal
ize
d to
are
a at
t=
0 h
t(h) t(h)
16
speciation differs in the respective three experiments. At pH 8.5, only the intensity of the
peaks changes during desorption, which indicates that the fast desorption is due to one surface
complex only (SC III in figure 9). At pH 5 and at the lower concentration, only small shifts
are observed in the spectra, and again only one surface complex is presumed to be involved
(SC II). At pH 5 and at the higher concentration, the surface species initially display the
highest wavenumber P-O mode. The desorption is also initially faster than at the lower
concentration. During desorption, however, the peak is red-shifted until the spectral features
converge with those collected at the lower concentration. The high frequency P-O mode is
consistent with SC I. Furthermore, in this surface complex the ligand is acting as a hydrogen
bond donor, and previous studies on polycarboxylates have shown that surface complexes
with hydrogen bond donor groups desorb faster than those with hydrogen bond acceptors.[33]
Figure 11. Left panel: Integrated absorbance between 1000 and 1230 cm-1
of G1P desorbing from goethite
at pH 8.5 and [G1P]tot = 1.36 µmol/m2, solid line represent the experimental data and the dotted line
describes a model fit assuming first order decay of one species. Right panel: calculated spectrum of the
kinetic component obtained from the model fit.
In addition to the qualitative analysis above, global fits using singular value decomposition
(SVD) were performed. The analysis of the spectra from the pH 8.5 experiment corroborated
the existence of one predominating surface complex. A kinetic model including a first order
decay of one species fits the experimental data (figure 11). The calculated spectrum of the
species is very similar to the spectrum collected at the start of the desorption experiment at pH
8.5 (figure 12). For the pH 5 data set, a three-species model with first order decay of two
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0 50 100 150 200
inte
gra
ted
ab
so
rba
nce
time/minutes
1230 1180 1130 1080 1030
ab
so
rba
nce
wavenumber/cm-1
17
species together with a third species growing in at a first order rate fits the experimental data.
The calculated spectra shown in figure 13 are in agreement with the expected features of SC I
– III. Spectra (a) and (c) are similar to the experimental spectra collected at the low and high
pH endpoints, displayed in figure 6. (c) is also very similar to the spectrum of the surface
complex at pH 8.5, while (a) is similar to the spectrum collected initially in the pH 5
experiment at the higher concentration. Spectrum (b) has P-O modes at wavenumbers in
between those of the other two complexes, and thus it is assigned to SCII. It also is similar to
the spectrum collected at the start of the desorption experiment at the lower concentration, as
well as spectra collected after some hours at the higher concentration. Spectra collected at the
end of the desorption experiment (not shown) are approaching the features of spectrum (c),
which corroborates the growth of this species.
Figure 12. Data from the desorption experiments a) initial spectrum at pH 5.0 and 1.36 µmol G1P/m2, b)
spectrum collected after 5 h at pH 5.0 and 1.36 µmol G1P/m2, c) initial spectrum at pH 5.0 and 0.69 µmol
G1P/m2, and d) initial spectrum at pH 8.5 and 1.36 µmol G1P/m
2.
1220 1170 1120 1070 1020
ab
so
rba
nce
wavenumber/cm-1
a
b
c
d
18
Figure 11. Left panel: Integrated absorbance between 1000 and 1230 cm-1
of G1P desorbing from goethite
at pH 5.0 and [G1P]tot = 1.36 µmol/m2, solid line represent the experimental data and the dotted line
describes a model fit assuming first order decay of two species and a third species growing in at a first
order rate. The predicted change of the kinetic components are denoted a, b and c. Right panel: calculated
spectra of the kinetic components obtained from the model fit. The labels correspond to the curves in the
left panel.
The kinetic behavior revealed in the SVD, with the decay of two species and the growth of a
third, shows that loss of a surface complex can have at least two causes: (1) desorption, and
(2) conversion into another surface species (which may be due to changes in surface charge as
a result of the changing coverage). Now the desorption process can be summarized in Scheme
1, which shows the difficulty in determining true desorption rates of individual surface
complexes.
Scheme 1. Possible scheme of reactions describing desorption of G1P from goethite at pH 5.
The desorption of SC III at pH 8.5 is considerably faster than the desorption at pH 5 of SC I
and SC II. Yet SC III grows in during the experiment at pH 5. This suggests that the rate of
desorption of an individual surface complex is pH dependent, which may be due to variations
in surface charge: SC III desorbs more slowly at pH 5 because the goethite surface is more
positive.
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0 200 400 600 800 1000
inte
gra
ted
ab
so
rba
nce
time/minutes
b
c
a
1230 1180 1130 1080 1030
ab
so
rba
nce
wavenumber/cm-1
a
b
c
SC I SC II SC III
kI
deskII
deskIII
des
kI-II
convkII-III
conv
19
5. Surface Promoted Hydrolysis of G1P and G6P Adsorbed onto Goethite
In pure ionic medium G6P and G1P were stable during 48 hours of monitoring, with glucose
concentrations remaining constant at 0.5-0.7 and 2 µM, respectively, regardless of pH (data
not shown). These negligible amounts may correspond to a glucose contamination in the G6P
and G1P salts used.
In presence of goethite, on the other hand, G1P and G6P show markedly different hydrolytic
behavior (figures 14 and 15). G6P predominantly hydrolyzes at low pH values, while G1P
hydrolyzes primarily at high pH values. Considering the stability of the compounds in pure
ionic medium, the glucose in the mineral containing samples is obviously the product of
surface-promoted hydrolysis. Since glucose has a weak affinity for the goethite surface, with
glucose adsorption increasing in an almost linear fashion with increasing pH (paper IV), the
glucose data is not expected to show the full extent of the hydrolysis. Nevertheless, the
different hydrolytic trends for G1P and G6P are undisputable.
G6P and G1P have the same leaving group, i.e. glucose, and the difference in hydrolytic
trends must thus be explained by other factors. Figure 16 compares the C-O-H bending
modes of the glucose group of adsorbed G1P and G6P. Small differences between the high
and low pH spectra in the G1P system indicate that the state of the glucose group/surface
interactions change with pH. On the other hand, pH has hardly any effect on the C-O-H
peaks, and hence the interactions, in the G6P system. Accordingly, the greater hydrolytic rate
at high pH values in the G1P system may partly be explained by increasing stabilization of the
leaving group, due to changes in hydrogen bond interactions.
In addition, the DFT-calculated structures of solvated G1P and G6P suggest that
electronic/bonding effects may influence the hydrolytic rate (paper III). According to the
calculations the P-OCX bond is 1.75 Å in G1P and 1.73 Å in G6P. The bond is thus slightly
weaker in G1P, and the lower electron density around the phosphorus atom in G1P makes it
the stronger electrophile. This explains the greater hydrolytic rate encountered in the G1P
system under basic conditions, under which the “hard” hydroxide ion acts as a nucleophile,
with a preference for the relatively “hard” phosphoryl center.
The nucleophilic attack, though, may also occur at the “soft” 1’ and 6’ carbon atom in G1P
and G6P, respectively, with a water molecule acting as the nucleophile.[34] Naturally, this
20
mechanism increases in importance as the pH decreases. Again, bond lengths can be invoked
to explain the greater hydrolytic rate displayed by G6P at low pH values. The calculated PO-
CX bond length is 1.41 Å in G6P and 1.38 Å in G1P, which indicates that the carbon atom in
G6P is the stronger electrophile.
Figure 14. Glucose concentration as a function of pH and time, with a total G1P concentration of a) 1.38
µmol/m2 of goethite, and b) 0.69 µmol/m
2 of goethite. and denotes 1 h samples, and 6 h samples,
and 24 h samples, and and 48 h samples. In c) the glucose concentrations are normalized to the
amount of G1P adsorbed. The error bars are based on the standard deviation from three individual
experiments.
0
2
4
6
8
10
12
2 3 4 5 6 7 8 9 10
µm
ol g
luco
se
pH
a)
0
2
4
6
8
10
12
2 3 4 5 6 7 8 9 10µ
mo
l g
luco
se
pH
b)
c)
0
0.5
1
1.5
2
2.5
3
3.5
4
2 3 4 5 6 7 8 9 10
Glu
co
se
(%
of
ad
so
rbe
d g
luco
se
-1-p
ho
sp
ha
te)
pH
µM
glu
cose
µM
glu
cose
21
Figure 15. Glucose concentration as a function of pH and time, with a total G6P concentration of (a) 1.36
µmol/m2 goethite and (b) 0.69 µmol/m
2 goethite: () 1 h samples, () 6 h samples, () 24 h samples, ()
48 h samples. (c) Glucose concentrations are normalized to the amount of G6P adsorbed (1.36 µmol
G6P/m2 goethite). The error bars are based on the standard deviation from three individual experiments.
Figure 16. Infrared spectra in the C-O-H bending region, of (a) G1P, and (b) G6P, adsorbed onto goethite.
The pH is 8.5 (thick line) and 5.0 (thin line). The lower spectra of weak intensities show the difference
between the pH 8.5 and 5.0 samples.
a) b)
c)
µM
glu
cose
Glu
cose
(%
of
adso
rbed
glu
cose
-6-p
ho
sph
ate)
µM
glu
cose
abso
rban
ce
wavenumber/cm-1
1520 1470 1420 1370 1320 1270 1220
ab
so
rba
nce
wavenumber/cm-1
abso
rban
ce
wavenumber/cm-1
a) b)
22
6. Enzymatic Hydrolysis of G1P and G6P Adsorbed onto Goethite
The enzymatic hydrolysis is discussed in papers II and III, for G1P and G6P, respectively.
AcPase hydrolyzes aqueous G6P faster than aqueous G1P (figure 17). A plateau of G6P
hydrolysis by the enzyme was reached after 3 to 6 hours where 85 to 90% of the added G6P
was hydrolyzed, compared to ca. 20% of the added G1P in the same period of time.
In presence of goethite, the hydrolytic rates decrease (figures 18 and 19). At 1.23 mM of
glucose phosphate, the hydrolytic rate is still on the same order of magnitude as in solution.
The rate approximately follows first-order kinetics, with the rate constants decreasing at pH
5.0 in the G6P system from 6.6*10-3
min-1
in solution to 1.2*10-3
min-1
at the interface. The
corresponding rates for G1P are 4.6*10-4
min-1
and 3.2*10-4
min-1
, respectively. At 0.64 mM
of ligand, the rate changes from 1.1*10-2
min-1
in solution to 9.0*10-5
min-1
at the interface for
G6P, and from 5.8*10-4
min-1
to 1.3*10-5
min-1
for G1P.
From figures 18 and 19, it is clear that the enzymatic hydrolysis is strongly dependent on the
total glucose phosphate concentration. Since practically all ligand is adsorbed in the pH
region 4.5 to 5.5, this implies a dependence on the surface coverage. As seen above,
desorption is slightly faster at the higher concentration, but the difference in desorption
kinetics alone cannot explain the large difference in rate.
Figure 17. Enzyme catalyzed release of glucose from G1P and G6P in aqueous solution at pH 5.0.
µM
glu
cose
t (h)
23
Figure 18. Enzyme catalyzed release of glucose from glucose-1-phosphate adsorbed on goethite.
Figure 19. Enzyme catalyzed release of glucose from glucose-6-phosphate adsorbed on goethite.
The enzymatic activity of AcPase decreases rapidly in the supernatant. At both ligand
concentrations, the remaining activity in the supernatant after one hour was below the
detection limit of the method (i.e. below 1.3 % of the initial activity), implying rapid
adsorption of the enzyme. In the infrared spectra from the G1P and G6P enzymatic
experiments, peaks are detected in the region 1500 – 1700 cm-1
(figures 20 and 24). These are
the amide I and II bands characteristic of proteins, confirming that AcPase adsorbs onto
goethite.
The following discussion focuses on G1P (paper II), but applies to G6P (paper III) as well.
Differences are pointed out at the end of the section.
µM
glu
cose
t (h)
µM
glu
cose
t (h)
24
Figure 20. Infrared spectra in the amide and G1P regions from the SIPT experiments of the reaction
between AcPase and G1P-goethite complexes at pH 5 and at 0.69 µmol G1P per m2 of goethite (A, B), 1.00
µmol G1P per m2 of goethite (C, D), 1.36 µmol G1P per m
2 of goethite (E, F). All spectra in the amide
region are plotted on the same scale and so are the G1P spectra. Note however the different ordinate
scales of the two regions. Each data set was collected over a 10 h period containing approximately 75
spectra. The arrows indicate intensity changes as a function of time.
The trends in the infrared spectra show that the hydrolytic rate is strongly dependent on the
total G1P concentration. At 0.69 μmol/m2 a small increase in the intensities of the G1P peaks
was observed. This is a consequence of the experimental setup: rapid G1P adsorption onto
particles close to the point of acid addition is followed by a slow redistribution onto the
goethite particles in the suspension and overlayer. The lack of amide I and II bands in these
spectra can be explained similarly: AcPase adsorbs rapidly onto high affinity sites on the
goethite particles at the point of addition, forming non-labile enzyme surface complexes. The
25
adsorption is irreversible, and since little or no redistribution occurs, the enzyme does not
adsorb onto the overlayer where the infrared measurements are made. These non-labile
surface complexes have a very low catalytical activity, as shown by the quantitative glucose
data (figure 18) and the peaks that grow faintly in the G1P-region of the infrared spectra.
At 1.00 μmol/m2, a slight decrease in the 1140 cm
-1 peak intensity is seen, which is consistent
with the increased hydrolysis at this concentration. In these spectra, the appearance of amide I
and II bands suggest that the enzyme is present in a more labile form that redistributes more
readily. This trend continues when the G1P concentration is increased to 1.36 μmol/m2, with a
clear decrease in the intensity of the 1140 cm-1
peak over time (figure 20 F), in accordance
with the dramatically increased hydrolytic rate. Figure 20 F shows another sign of hydrolysis
in the form of an increased intensity at 1042 and 1095 cm-1
. These peaks are practically
identical to those of orthophosphate adsorbed onto goethite (paper II). Concomitantly, the
amide I and II bands increased noticeably, suggesting either an even more labile enzyme
surface complex, or an increase of the labile fraction.
Figure 21. The fraction of hydrolyzed G1P-goethite complexes at pH 5 and 1.36 µmol G1P per m2 of
goethite calculated from the measured glucose concentrations in solution (red squares), and from the main
IR band at 1140 cm-1
of adsorbed G1P (blue diamonds). The amide band area is the integrated area
between 1450 and 1700 cm-1
(green triangles).
Figure 21 displays the fraction of hydrolyzed G1P with higher surface coverage at pH 5.0,
estimated from the measured glucose concentration, and from the ratio between the 1140 cm-1
intensities at t0 (i.e. when enzyme is added) and t. Also displayed are the integrated areas of
the amide I and II bands. Figure 21 shows that the accumulation of AcPase on the goethite
overlayer (which represents the fraction of enzyme that was rapidly distributed over all
0
0.05
0.1
0.15
0.2
0.25
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8 10
Amideban
darea
Frac
onofhyd
rolyzedG1P
Hours
26
particles in the system) was initially faster than the rate of hydrolysis. This is in agreement
with the 2D correlational analysis of the spectra, displayed in figure 22. The negative peaks at
(1148, 1548) and (1148, 1638) in the synchronous map shows that the intensities of the amide
I and II bands change in opposite direction to the intensity of the G1P peak. These intensity
changes are coincidental. However, corresponding peaks in the asynchronous map show that
the changes occur slightly out-of-phase. According to Noda’s rule (see 1.4), the positive
peaks at (1148, 1548) and (1148, 1638) in the asynchronous map indicates that the amide I
and II bands change before the G1P peak. This shows that a build-up of a catalytically active
surface layer of AcPase is necessary for hydrolysis.
Figure 22. Synchronous (left) and asynchronous (right) contour plots obtained from the heterogeneous 2D
correlation spectroscopy analysis of the infrared spectra presented in Figs. 20 E and F. Grey areas show
negative regions, and white areas positive.
Previously, different enzyme adsorption modes were suggested, depending on the G1P
surface coverage. A closer look at the bands in the amide I and II region give further
indication of different modes. Compared to the spectrum of aqueous AcPase, the amide I band
on pure goethite and initially at 1.00 μmol of G1P/m2 is broadened and blue-shifted (figure
23). Also, the amide II band is very broad. This suggests structural distortions of the enzyme.
With time the bands shift to the band positions in aqueous AcPase, but remain broad. At 1.36
μmol/m2 the spectra initially is in agreement with that of the aqueous enzyme, but over time
band broadening occurs, indicating distortions.
wav
en
um
be
r/cm
-1
wav
en
um
be
r/cm
-1
wavenumber/cm-1 wavenumber/cm-1
27
Figure 23. Normalized infrared spectra in the amide region from the SIPT experiments of the reaction
between AcPase and G1P-goethite complexes at pH 5.0 and 1.00 µmol G1P per m2 of goethite (left), and
1.36 µmol G1P per m2 of goethite (right). For comparison infrared spectra of AcPase adsorbed to goethite
(a) and an aqueous solution of AcPase (b) have been included in the left panel. The spectrum of the
aqueous solution of AcPase is also included in the right panel (red spectrum drawn with thicker line
width). The arrows indicate directions of shifts as a function of time.
The enzyme becomes substantially distorted when adsorbing onto the positively charged
goethite surface at low ligand surface coverage (or in absence of ligand). When the structure
of the enzyme is distorted, the catalytic activity is lost. At a high ligand concentration the G1P
surface complexes block the high-affinity sites. In addition, due to the glucose moieties,
adsorbed G1P gives the goethite surface organic-like properties. The modified surface is
expected to have a hydrophobic character, as well as different hydrogen bonding properties
compared to pure goethite. In this case the interactions between AcPase and the surface do not
distort the enzymatic structure, and the enzymatic activity is retained.
1425147515251575162516751725
Absorban
ce
Wavenumber/cm-1
1425147515251575162516751725
Absorban
ce
Wavenumber/cm-1
28
Figure 24. Infrared spectra in the amide and G6P regions from the SIPT experiments of the reaction
between AcPase and G6P-goethite complexes at pH 5 and at 0.69 µmol G6P per m2 of goethite (A, B), and
at 1.36 µmol G6P per m2 of goethite (C, D). All spectra in the amide region are plotted on the same scale
and so are the G6P spectra. Note however the different ordinate scales of the two regions. Each data set
was collected over a 10 h period containing approximately 75 spectra. The arrows indicate intensity
changes as a function of time.
As mentioned, the AcPase hydrolysis of G6P surface complexes (paper III) is similar to that
observed in the corresponding G1P system (paper II). However, there are some notable
differences. At the low surface coverage in the G1P system, no amide bands were observed,
indicating very non-labile AcPase-goethite surface species and a slow, if any, redistribution
process. On the other hand, spectra in the amide region collected at the low G6P surface
coverage are similar to those collected at 1.00 µmol G1P/m2 (figures 24 A and 20 C). The fact
that significant amide bands are detected at the low G6P surface coverage, suggests that the
properties of the surface layers created by the two different glucose phosphates are somewhat
abso
rban
ce
abso
rban
ce
abso
rban
ce
abso
rban
ce
wavenumber/cm-1wavenumber/cm-1
wavenumber/cm-1wavenumber/cm-1
A
DC
B
29
different, with G6P facilitating the formation of relatively more labile AcPase. This can be
related to the discussion on the abiotic hydrolysis where infrared data indicated differences
between G1P and G6P with respect to the interactions between the glucose moiety and the
surfaces (figure 16). Therefore, the conformations of the G6P surface complexes may
relatively favour the formation of labile AcPase surface species. The presence of more labile,
and hence more catalytically active, AcPase in the G6P system compared to G1P at low
ligand concentrations is in agreement with the higher rate of hydrolysis in the former. The
rate constants in these systems are 9.0*10-5
min-1
and 1.3*10-5
min-1
, respectively.
In solution the G1P/G6P ratios of the rate constants were 4.56*10-4
/6.6*10-3
= 0.07 (1.23
mM) and 5.75*10-4
/1.1*10-2
= 0.05 (0.69 mM) whereas the corresponding ratios for the
surface hydrolysis were 3.2*10-4
/1.2*10-3
= 0.27 (1.23 mM) and 1.3*10-5
/9.0*10-5
= 0.14.
According to the ratios the greater specificity of AcPase for G6P is partly lost at the surface. It
is possible that the structural properties that create the specificity of AcPase are sensitive to
small distortions. Furthermore, other surface processes may control the overall hydrolysis
rates such as desorption and surface diffusion, and can thus be part of the explanation as to
why the rates of the G1P and G6P hydrolysis are more similar on goethite than in solution.
PART II Adsorption mechanisms of glucose in aqueous goethite
suspensions
1. Introduction
Glucose adsorption is of interest to many areas of research, such as soil science and
engineering. For example, it has been shown that surface interactions may reduce the
bioavailability or alter the reaction pathways of glucose.[35, 36] The actual adsorption
mechanisms have mainly been studied in the area of mineral processing. Since the 1930s
polysaccharides have been used in the mining industry as depressants in flotation
processes.[37] In order to better understand these processes, a number of studies have been
devoted to the mechanisms of glucose and polysaccharide adsorption onto mineral surfaces.
In early studies hydrogen bonding was presumed to be the main attractive force.[38] Later it
was suggested that hydrophobic effects were provided additional stability to the surface
complexes.[39] Recent studies, however, have partly dismissed these propositions. The
30
current view, based on results from infrared spectroscopy, is that glucose’s oxygen atoms
interact directly with the surface sites on the mineral, forming inner sphere complexes.[40-42]
2. Adsorption of Glucose on Goethite
In paper IV the adsorption mechanisms of glucose in aqueous goethite suspensions is
discussed. Glucose has a pKa of approximately 12.1. Therefore, glucose is uncharged under
natural conditions, and has markedly different adsorption characteristics compared to the
anionic G1P and G6P. Firstly, glucose adsorption is not extensive. Secondly, adsorption of
anions onto goethite decreases with increasing pH, as a result of decreasing surface charge
and competition from hydroxide ions. In contrast, figure 25 shows how glucose adsorption
increases with pH, in an almost linear fashion. The predicted pH dependent concentration of
deprotonated surface sites resembles this curve, suggesting that they are involved in the
process.[43]
Figure 25. Adsorption of glucose as a function of pH, with a total glucose concentration of 0.95 µmol/m2 of
goethite. The error bars are based on the standard deviation from two individual experiments.
3. FTIR Characterization of Glucose Adsorbed onto Goethite
The infrared spectrum of aqueous glucose is displayed in figure 26. The assignment of the
major peaks (table 3) is based on the work by Suzuki and Sota,[44] and DFT calculations
made for this study. The normal vibrational modes of glucose are very complex, and in the
DFT calculations only one conformation was examined. Therefore, the assignment describes
only the main features of the modes. The peaks (apart from the one at 1202 cm-1
) in the
nm
ol a
ds.
/m2
% ad
s.
pH
31
depicted section of the aqueous spectrum originate from modes containing substantial
contributions from C-O stretching motions (νC-O).
Figure 26. Left panel: Infrared spectra of glucose adsorbed on goethite at a) pH 10.29, b) pH 9.82, c) pH
8.92, d) pH 8.31, e) pH 7.11, f) pH 6.11, g) pH 5.27, h) pH 5.08. Spectrum i) is a 50 mM aqueous solution of
glucose at pH 6, j) is a hydrated goethite surface at pH 6, and k) is of a 1 mM aqueous solution of glucose
at pH 6. Right panel: a blow-up of the infrared spectra of glucose adsorbed on goethite with labels
corresponding to those in the left panel. The arrows highlight the νC1-O5+νC5-O5 peaks of α-glucose (1058
cm-1
) and β-glucose (1080 cm-1
).
Table 3. Assignment of the main features of the normal modes of D-glucose in aqueous solution.
Wavenumber/cm-1
Assignment
1035 νC1-O5+νC2-O2+νC6-O6
1058a νC1-O5+νC5-O5
1080b νC1-O5+νC5-O5
1107 νC-Oc
1150 νC1-O1+νC-C
1202 δCH+δCOH
aα-glucose;
bβ-glucose;
cmode involving C-O motions of practically all groups
1250 1200 1150 1100 1050 1000
wavenumber/cm-1
a
b
c
d
e
f
g
h
i
j
k
1200 1180 1160
wavenumber/cm-1
a
b
c
d
e
f
g
h
C1
O1
C2
O2
C3O3
C4
O4
C5
O5
C6
O6
32
When glucose adsorbs onto goethite, the glucose ring remains intact. This is evident from the
peaks at 1035 and 1080 cm-1
, which originate primarily from νC1-O5 and νC5-O5 (figure 26).
Furthermore, glucose at the goethite surface is predominantly found in the β-form: the 1058
cm-1
peak of the α-form is not seen in the spectra of the adsorbed species.
Adsorption clearly changes at least some of the C-O bonds. The 1150 cm-1
peak, originating
to a large extent from νC1-O1, is shifted to 1175 cm-1
in the adsorbed spectra, and is further
blue-shifted as the pH increases. Less conspicuous differences from the solution spectrum
indicating C-O bond alterations are broadening and the development of shoulders of the 1107
cm-1
peak, and an intensity increase at 1202 cm-1
.
A series of DFT calculations with differing numbers of explicit water molecules show how
strongly hydrogen bonding affects the vibrational frequencies of aqueous glucose (figure 27).
Shifts of the 1150 cm-1
band in the calculated spectra, induced by varying numbers of water
molecules, correlates roughly to the C1-O1 bond lengths. The bond lengths in turn correlate to
the hydrogen bond strength: increasing the hydrogen bond strength when glucose acts as a
donor lengthens the C1O1-H bond and simultaneously shortens the C1-O1H bond; in infrared
spectra this is observed as a blue-shift of the νC1-O1 peak.
Figure 27. Left panel: DFT calculated infrared spectra of β-glucose in chair conformation a) in gas phase,
b) with 1 explicit water molecule, c) 2 explicit water molecules, d) 4 explicit water molecules, e) 6 explicit
water molecules, f) 8 explicit water molecules, g) 10 explicit water molecules. The experimental infrared
spectrum of aqueous glucose is shown in h). The DFT calculated spectra were simulated assuming
Lorentzian line shape and a full-width-at-half-maximum-height of 15 cm-1
. Right panel: Vibrational
frequencies of the νC1-O1 mode as a function of the C1-O1 bond distance.
1200 1150 1100 1050 1000
wavenumber/cm-1
a
b
c
d
e
f
g
h1122
1127
1132
1137
1142
1147
1.37 1.374 1.378 1.382 1.386
wa
ve
nu
mb
er/
cm
-1
C1-O distance/Å
33
As shown by the DFT calculations, small changes in bond length can have a large effect on
vibrational frequencies (figure 27). Thus, the 25 cm-1
blue-shift of the νC1-O1 peak observed
when glucose adsorbs onto goethite may indicate that the C1O1H group acts as a hydrogen
bond donor towards the acceptor sites on the surface. The most acidic surface sites are
presumably also the weakest hydrogen bond acceptors, hence as the pH increases,
deprotonation of increasingly less acidic sites forms acceptor sites with increasing strength.
This explains the pH dependent blue-shift seen in experimental spectra. As noted above, the
concentration of deprotonated (i.e. hydrogen bond acceptor) sites increases linearly with pH,
resulting in the near linear adsorption curve in figure 25.
The case against inner sphere complexation becomes stronger when taking the weak peaks in
the 1300-1500 cm-1
region into account. The peaks here mainly originate from COH bending
modes (δCOH), and they differ from the spectrum of aqueous glucose as well as showing pH
dependence (figure 28). The ratio between the total absorbance of the COH region to that of
the region 1000 - 1200 cm-1
is roughly the same for both the aqueous glucose and the surface
complexes. This shows that little if any deprotonation occurs in the COH group, since
otherwise the intensity in the 1300-1500 cm-1
region would decrease. An inner sphere
complex would require deprotonation of at least two of the five hydroxyl groups of glucose.
Figure 28. Infrared spectra of glucose adsorbed on goethite at a) pH 5.08, b) pH 5.27, c) pH 6.11, d) pH
7.11, e) pH 8.31, f) pH 8.92, g) pH 9.82, h) pH 10.29. Spectrum i) is a 50 mM aqueous solution of glucose at
pH 6.
1520 1470 1420 1370 1320
ab
so
rba
nce
wavenumber/cm-1
a
b
c
d
e
f
g
h
i
34
Hydrogen bonding is also suggested via the shift of the peak in the COH region when glucose
adsorbs onto goethite. Similar effects have been reported when carboxylic acid groups act as
hydrogen bonding donors.[31, 32] The peak at 1420 cm-1
broadens with increasing pH, and
this broadening is correlated to the blue-shift of the νC1-O1H peak. The two pH-dependent
effects may be caused by increasing hydrogen bond interactions between the C1O1H group
and acceptor sites.
Conclusions
In this study, a technique for simultaneous infrared and potentiometric titrations (SIPT)
marked a new approach to the molecular-level study of enzymatic processes. The possibility
of in-situ FTIR measurements of enzymatic reactions at the mineral/water interface will likely
be of great use in future studies. Questions suitably approached with the SIPT-technique are
for example those raised in the present study regarding enzyme lability and its connection to
catalytic activity. Future studies may include other enzymatic systems, such as those of
importance for the C-cycle.
In this dissertation, it was found that AcPase, like G1P and G6P, adsorbs onto goethite. Thus
the surfaces of environmental particles can serve to bring substrates and enzymes together,
creating micro-environments of high enzymatic activity. A central finding is that the
enzymatic hydrolysis is strictly an interfacial process. Consequently, measurements of
enzymatic activity in soil solutions likely underestimate the overall activity in soils.
Furthermore, the surface properties of environmental particles can have a strong effect on the
adsorption mode of enzymes, thus affecting their activity. At low glucose phosphate
coverage, the enzyme interacts strongly with the surface, forming non-labile enzyme surface
complexes. Distortion of the enzyme surface complex leads to a considerable decrease in
hydrolytic activity. At a higher ligand concentration the high-affinity sites on goethite are
covered by glucose phosphate. The glucose moieties lend the surface organic-like properties,
leading to weaker interactions with the enzyme. As a result, the structure of the enzyme is less
distorted and the hydrolytic rate is greater.
35
References
[1] P. Hinsinger, Plant and Soil 237 (2001) 173.
[2] A. Paytan, K. McLaughlin, Chem. Rev. 107 (2007) 563.
[3] T. Hiemstra, W.H. VanRiemsdijk, Journal of Colloid and Interface Science 179 (1996)
488.
[4] N. Khare, D. Hesterberg, J.D. Martin, Environ. Sci. Technol. 39 (2005) 2152.
[5] W. Yao, F.J. Millero, Environ. Sci. Technol. 30 (1996) 536.
[6] E.A. Paul, F.E. Clark, Soil microbiology and biochemistry. 2 ed., Academic Press, New
York, NY, 1996.
[7] D. Bourke, P. Dowding, H. Tunney, J.E. O'Brien, D.W. Jeffrey, Biol. Environ.-Proc. R.
Irish Acad. 108B (2008) 17.
[8] R.J. Chrost, Environment control of the synthesis and activity of aquatic microbial
ectoenzymes, Springer, 1991, p 29.
[9] A. Torrents, A.T. Stone, Soil Sci. Soc. Am. J. 58 (1994) 738.
[10] A. Dannenberg, S.O. Pehkonen, Journal of Agricultural and Food Chemistry 46 (1998)
325.
[11] D.S. Baldwin, J.K. Beattie, L.M. Coleman, D.R. Jones, Environmental Science &
Technology 29 (1995) 1706.
[12] D.S. Baldwin, A.K. Beattie, L.M. Coleman, Environmental Science & Technology 35
(2001) 713.
[13] H.W. Luo, R. Bennera, R.A. Long, J.J. Hu, Proc. Natl. Acad. Sci. U. S. A. 106 (2009)
21219.
[14] F. Leprince, H. Quiquampoix, Eur. J. Soil Sci. 47 (1996) 511.
[15] C. Giaveno, L. Celi, A.E. Richardson, R.J. Simpson, E. Barberis, Soil Biology &
Biochemistry 42 (2010) 491.
[16] P. Nannipieri, P. Sequi, P. Fusi, Humus and enzyme activity, Elsevier, 1996, p 293.
[17] A. Naidja, P.M. Huang, J.M. Bollag, J. Environ. Qual. 29 (2000) 677.
[18] H.K. Pant, P.R. Warman, J. Nowak, Commun. Soil Sci. Plant Anal. 30 (1999) 757.
[19] M. Espinosa, B.L. Turner, P.M. Haygarth, J. Environ. Qual. 28 (1999) 1497.
[20] M.A. Ali, D.A. Dzombak, Environ. Sci. Technol. 30 (1996) 1061.
[21] M. Kruzel, B. Morawiecka, Acta Biochim. Pol. 29 (1982) 321.
[22] Y. Sugiura, H. Kawabe, H. Tanaka, S. Fujimoto, A. Ohara, J Biol Chem 256 (1981)
10664.
[23] D.C. Harris, Quantitative chemical analysis. 6 ed., W.H. Freeman, New York, 2002.
36
[24] J.S. Loring, M.H. Sandstrom, K. Noren, P. Persson, Chem.-Eur. J. 15 (2009) 5063.
[25] T. Wei, S. Kaewtathip, K. Shing, J. Phys. Chem. C 113 (2009) 2053.
[26] I. Noda, Y. Ozaki, Two-dimensional correlation spectroscopy : applications in
vibrational and optical spectroscopy John Wiley & Sons, Chichester, West Sussex,
England, 2004.
[27] W. Stumm, Chemistry of the solid-water interface: processes at the mineral-water and
particle-water interface in natural systems. Wiley, New York, 1992.
[28] G. Sposito, The surface chemistry of natural particles. Oxford University Press, New
York, 2004.
[29] M.I. Tejedortejedor, M.A. Anderson, Langmuir 6 (1990) 602.
[30] P. Persson, N. Nilsson, S. Sjoberg, Journal of Colloid and Interface Science 177 (1996)
263.
[31] M. Lindegren, P. Persson, Eur. J. Soil Sci. 60 (2009) 982.
[32] M. Lindegren, P. Persson, Journal of Colloid and Interface Science 343 (2010) 263.
[33] M. Lindegren. Aqueous Surface Chemistry of Goethite-Adsorption and Desorption
Reactions Involving Phosphate and Carboxylic Acids. Doctoral Thesis, Umeå
University, Umeå, 2009.
[34] P. Hendry, A.M. Sargeson, Prog. Inorg. Chem. 38 (1990) 201.
[35] D.L. Jones, A.C. Edwards, Soil Biol. Biochem. 30 (1998) 1895.
[36] R. Jandl, R.S. Sletten, J. Plant Nutr. Soil Sci.-Z. Pflanzenernahr. Bodenkd. 162 (1999)
623.
[37] M.C. Fuerstenau, G. Jameson, R.-H. Yoon, Froth flotation: a Century of innovation.
SME, Littleton, 2007.
[38] S.R. Balajee, I. Iwasaki, Transactions of the Society of mining engineers of AIME 244
(1969) 401.
[39] J.M. Wie, D.W. Fuerstenau, International Journal of Mineral Processing 1 (1974) 17.
[40] P.K. Weissenborn, L.J. Warren, J.G. Dunn, Colloid Surf. A-Physicochem. Eng. Asp. 99
(1995) 11.
[41] Q. Liu, Y.H. Zhang, J.S. Laskowski, International Journal of Mineral Processing 60
(2000) 229.
[42] J.S. Laskowski, Q. Liu, C.T. O'Connor, International Journal of Mineral Processing 84
(2007) 59.
[43] J.F. Boily, J. Lutzenkirchen, O. Balmes, J. Beattie, S. Sjoberg, Colloid Surf. A-
Physicochem. Eng. Asp. 179 (2001) 11.
[44] T. Suzuki, T. Sota, J. Chem. Phys. 119 (2003) 10133.
37
Acknowledgements
Tack till mina handledare Per Persson och Reiner Giesler, som delat med sig av både
kunskap och entusiasm. Och till John Loring och András Gorzsás för deras arbete med
Sally the SIPT. Tack också till alla på plan 6 som gör korridoren till en fantastisk arbetsplats.
Särskilt tack till rumskompisarna Hanna och Ola, samt till Janice och Malin för vänskap
under den här tiden.
Sist men inte minst vill jag tacka min Malin för allt stöd och alla äventyr, forna och
kommande.