Biochimica et Biophysica Acta 1774 (2007) 233 – 242

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Compensatory secondary structure alterations in protein glycation

Ranjita GhoshMoulick a , Jaydeep Bhattacharya a , Shibsekhar Roy a ,
Soumen Basak b , Anjan Kr. Dasgupta a,⁎
a
Department of Biochemistry, Calcutta University, 35 Ballygunge Circular Road, Calcutta 700019, India
b
Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Calcutta 700064, India
Received 19 June 2006; received in revised form 25 November 2006; accepted 30 November 2006
Available online 13 December 2006

Abstract

Glycation, a local covalent interaction, leads to alterations in secondary and tertiary structures of hemoglobin, the changes produced by fructose
being more pronounced than those caused by glucose. The Stokes diameter of hemoglobin increases upon glycation from 7 to 14 nm and a
concurrent inter-chain cross-linking and heme loss are also observed, particularly in the later stage of glycation. An initial increase of tryptophan
(trp) fluorescence was observed in both glucation and fructation. In case of frucation however there was a decrease in tryptophan fluorescence that
was accompanied by an increase in fluorescence of the advanced glycosylation end products (AGEs). This fluorescence behavior is indicative of
energy transfer between tryptophan and the AGEs formed during the late stage of glycation. Emergence of an isosbestic point in the fluorescence
spectra (taken at different time intervals) implies existence of two distinct glycation stages. The late glycation stage is also marked by an increase
of beta structure and random coil at the expense of alpha helix. It is further observed that this compensatory loss of alpha helix (reported for the
first time) and increase in beta sheet and random coil elements depend on the number of solvent-accessible glycation sites (rather than total
number of such sites) and the subunit assembly of the protein.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Glycation; Cross-Linking; Fluorescence; Circular Dichroism; Alpha helix; Beta sheet

1. Introduction diabetes is not dependent on sequence but is a result of direct

Methods for the detection of glycation-induced changes
in proteins can be broadly divided into two categories. Firstly
one detects the products of glycosylation reactions, e.g. ad-
vanced glycosylated end products (AGEs). Secondly, changes
in either protein or sugar structure using spectroscopic methods
such as circular dichroism [1] or infrared spectroscopy [2] are
monitored.
Glycation is particularly important in diabetes, which has
been recently termed as a conformational disease [3].
Conformational diseases are conditions that arise from the
dysfunctional aggregation of proteins in non-native conforma-
tions. Unlike the usual amyloid peptides, whose amino acid
sequence thermodynamically drives them to have a higher
propensity of β-structure, the conformational aberration in
⁎ Corresponding author.
E-mail address: adgcal@gmail.com (A.K. Dasgupta).
1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2006.11.018
and indirect stresses produced as a result of poor control of
sugar. Exposure to high concentrations of sugar leads to
formation of a reversible Schiff's base and is followed by
emergence of covalently bonded adducts e.g. the AGE peptides
[4], which in turn leads to a number of pathological effects [5].
Importantly, these adducts can form only at specific sites where
lysine (or arginine) is present. Such local distortions may have
an impact on the overall structural milieu of the protein, such as
a rise in β-structure, leading to protein aggregation [6].
There are two major questions regarding the conformational
changes that may be associated with disease. First, it is
important to know the possible effects of the change of
conformation so that better structural insights into the
pathogenesis of the disease are obtained. For example, the
high loss of heme from glycated hemoglobin known to affect
diabetes patients [7,8] may imply that the structural integrity of
the assembly of α- and β-chains in hemoglobin is impaired, as
heme helps to maintain such integrity. For non-heme proteins,
234 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
glycation-induced overall changes in conformation have already
been reported [1]. For human serum albumin (HSA), glycation
results in a higher propensity for formation of β-sheet. In this
report we have shown that β-sheet generation is accompanied
by loss of alpha helix in a compensatory way. Furthermore,
the kinetics of such process is faster in case of hemoglobin as
compared to HSA. The loss of heme associated with glycation,
and the protein aggregation that follows could possibly be an
effect rather than a cause of the overall change in structure.
The other question that is equally important is how to
quantify such complex changes. There is an inherent difficulty
in quantifying the detailed structural changes associated with
glycation. As recently reported [2], the presence of sugar
complicates the determination of protein structures by conven-
tional techniques like X-ray crystallography. The difficulty of
obtaining enzymatically glycosylated proteins is well known
[9]. As far as the non-enzymatic glycation is concerned, to our
knowledge till today there is no entry in the protein databank.
The authors [2] used IR spectroscopic techniques to monitor
alterations in the sugar moiety in glycosylated proteins. Solving
structures of glycated proteins by NMR is also problematic as
the increase in rotational diffusion causes a line-broadening
effect. However NMR studies have been made with small
glycated peptides [10]. The search for simple and reliable new
techniques to assay glycation is equally important for a better
understanding of the steps involved in this important post-
translational event that is amplified in diabetes.
The present paper clearly demonstrates the two steps in
glycation, namely Schiff's base formation and formation of the
Advanced Glycosylation End products (AGEs) [11]. The steps
can be clearly distinguished from the biphasic time profile of
tryptophan fluorescence. In the initial phase the unfolding effect
dominates leading to gradual increase in tryptophan fluores-
cence. In the second phase, there is higher propensity for
formation of β-structure (at the expense of α-helix), leading to
possible protein aggregation.
2. Experimental
2.1. Sample preparation
Blood samples were collected in EDTA vials from normal healthy persons.
The whole blood was centrifuged at 3000×g for 10 min. and washed 3 times in
normal saline. The red cells were finally lysed by water. Toluene was used to
separate the RBC membrane from the liquid phase during lysis. The solution
was applied to a Sephadex G-50 column equilibrated with 0.1 M phosphate
buffer pH 7.0. Elution was carried out with 0.1 M phosphate buffer pH 7.0 at a
flow rate of 25 ml/h. The spectral purity of the preparation was checked by
comparing the Soret peak, as well as the peak at 280 nm, with those of human
hemoglobin (HbA0) procured from Sigma.
The other two proteins, HSA (Human Serum Albumin, A-9511) and
lysozyme (124013) were procured from Sigma and SRL respectively.
2.2. Preparation of glycated proteins
Hb, HSA and lysozyme stock solutions were prepared in 0.1 M phosphate
buffer of pH 7.2. For the fructose dependent kinetics experiments, all the
proteins (Hb, HSA and lysozyme) were incubated in 0.05 M fructose at 37 °C.
The final concentration of each protein in the incubation mixture at the start of
the reaction was 1 mg/ml. All solutions were prepared under sterile condition
using laminar flow to avoid contamination (fungal or bacterial). Aliquots were
withdrawn each day from the reaction mixtures for further studies. For the
experiments comparing the effects of different sugars, the Hb solution was
incubated in various concentrations of glucose and fructose (0.1–0.5 M at 27 °C)
for 30 days to get glycated and fructated Hb, respectively.
2.3. Fluorescence assay of glycation related structural changes and
formation of AGEs
Aliquots (∼ 1 μM) from glycated or fructated proteins prepared by
incubation with glucose (0.05 M) or fructose (0.05 M) were aseptically
withdrawn each day for the kinetics experiment for measurement of fluorescence
emission from the protein or from AGEs. The periodate method [12] matched
well with the initial rise of glycation but was unsuitable for fructosylation assay
[13]. Formation of AGEs was assayed fluorometrically as described in [13] by
measuring the fluorescence emission at 450 nm, using excitation at 350 nm. For
the experiment to compare the effect of different sugars (fructose and glucose)
the aliquots were withdrawn after 30 days for measurement of AGEs. The
durations of incubation by glucose and fructose were the same but the amount of
AGE products formed by the respective sugars varied considerably. Formation
of the two AGE products, pentosidine and malondialdehyde (MDA), was also
assayed by exciting the samples at 335 and 370 mm, respectively [14,15]. All
fluorescence measurements were made on a Hitachi F3010 spectrofluorometer.
Trp fluorescence [16] was measured using 280 nm excitation and excitation/
emission bandwidths of 5 nm each.
2.4. Gel electrophoresis
11% native gel and 12% SDS-PAGE were prepared to run the glycated/
fructated Hb, prepared by incubating Hb with different concentrations of
glucose/fructose for 30 days at 27 °C. For kinetics study in SDS-PAGE,
fructated Hb samples were prepared by incubating Hb in 0.05 M fructose at
37 °C. The samples were withdrawn each day from the respective reaction
mixtures and were loaded (kinetics). The purity of the protein was also
confirmed by SDS-PAGE, a single monomeric band appearing in case of
Hb whereas band multiplicity was observed for glycated or fructated
samples.
Fructosylated HSA and lysozyme were also monitored in SDS-PAGE.
Aliquots from the reaction mixture of the proteins and 0.05 M fructose were
pulled on the 0th, 3rd and 6th day for analysis.
2.5. Circular dichroism (CD) studies
CD spectra were recorded on a Jasco J720 spectropolarimeter at 25 °C. Two
sets of wavelength ranges, namely 190–250 nm and 250–500 nm, were chosen
for these studies. The 190–250 nm range was used to monitor the secondary
structure. The other range (250–500 nm), that spans the Soret region, was useful
in detecting the tertiary structural changes cause by loss of heme. Protein
samples, at a concentration of 0.1 mg/ml, were placed in cylindrical quartz
cuvettes of path length 1 mm. All spectra shown are averages of 5 scans, with the
far-UV spectra recorded at a scan rate of 20 nm/min and the near-UV ones
recorded at 50 nm/min. Spectra were corrected by subtracting appropriate buffer
blanks and smoothed by noise reduction.
CD measurements were performed on Hb samples withdrawn every 24 h for
1 week from the reaction mixture of 0.05 M fructose and Hb (kept at 37 °C). The
measurements were repeated on another set of Hb samples incubated at 40 °C.
The measured spectra were resolved into components representing different
types of secondary structure (helix, strand, random coil etc.) and their relative
percentages calculated [17] using the program CONTIN [18] from the suite of
programs available at the online site DICHROWEB (http://www.cryst.bbk.ac.
uk/cdweb/). The results of such analysis of our control Hb sample matched well
with those reported for the CD data of a reference Hb sample [19]. The
secondary structure estimation was also repeated using the program K2D
[20,21] available at DICHROWEB and the results compared with those found
using CONTIN (Fig. 7). A small variation in temperature (Fig. Sm3) did not
change the complementary nature of the α-helix to β-sheet conversion of the
protein.
 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
Samples for measurement of CD spectra of HSA and lysozyme were
withdrawn on the 6th day of the fructosylation reaction (initiated by incubation
in 0.05 M fructose at 37 °C). Spectra of the untreated proteins (control) were also
measured.
2.6. Heme loss associated with glucation and fructation
Absorbance spectra were measured on a Spekol 1200 (Analytik Jena) UV-
visible absorption spectophotometer using a quartz cuvette of path length 1 cm.
The kinetics of heme release were monitored by measuring the absorbance
spectrum of fructated Hb, prepared as mentioned above. Aliquots were pulled on
a daily basis, from 0th to 6th day. The average of three independent
measurements of the absorbance at the Soret peak (415 nm) was recorded as
a function of the number of days. Absorption spectra were also recorded for
samples of glucated and fructated Hb incubated for 30 days in varying
concentrations of sugars to know the status of the heme (Fig. 4A).
2.7. Dynamic light scattering (DLS)
A Nano-ZS (Malvern) instrument (5 mW He–Ne laser, λ = 632 nm) was used
for this purpose. The sample was poured in a DTS0112-low volume (1.5 ml)
disposable sizing cuvette of path length 1 cm. Prior to the study, protein samples
(typical concentration ∼ 10 μg/ml) were passed through a 2 μm filter. The
operating procedure was programmed (using the DTS software supplied with the
instrument) to record the average of 25 runs, each run being collected for 15 s,
with equilibration time of 3 min. The temperature was fixed at 25 °C. A particular
hydrated diameter (dh) was evaluated several times and the result presented in
terms of a distribution of the hydration diameter. The diameters of glycated and
fructated Hb was measured by DLS after incubating the samples for 30 days at
27 °C in presence of 0.5 M each of glucose and fructose, respectively.
3. Results
3.1. Glycation induced enhancement of hydrodynamic size
Glycation or fructation of hemoglobin (Hb) leads to an
increase in hydrodynamic size of the protein. To study the
dependence of this effect on the type of sugar or the length of
incubation period, we compared the size distribution of glycated
(glucose mediated) or fructosylated (fructose mediated) samples.
The results of four independent measurements were subjected to
one-way ANOVA test and are represented by a box plot (Fig. 1).
The mean hydrodynamic diameter (dh) increased from 7 nm for
untreated hemoglobin (control) to 11.5 nm and 10.5 nm for
hemoglobin incubated in 0.5 M each of glucose and fructose,
respectively. The p-value of the control, when compared to
glycated (GHb) or fructated (FHb) hemoglobin separately and
both together, was 0.006, 0.0009 and 0.003, respectively,
indicating a significant difference in the size (dh). The p-value
increased to 0.42 if only GHb and FHb were considered. This
implied that changes in size produced by the two sugars are not
significantly different. To summarize, glycation due to fructose or
glucose produces similar and appreciable increases in the
hydrodynamic size (dh) of hemoglobin.
3.2. AGE accumulation and intrinsic protein fluorescence
The fluorescence spectra of hemoglobin during the fructosyla-
tion reaction shown in Fig. 2 indicate changes in protein structure
due to reaction with fructose (0.05 M at 37 °C, incubation
temperature raised to achieve faster glycation kinetics). The
235
Fig. 1. Box plot for hydrodynamic diameter (dh) of normal Hb and Hb incubated
for 30 days in 0.5 M of glucose and fructose, respectively at 27 °C, as determined
using dynamic light scattering. Values shown are averages for 10–15 samples
each. Fructation and glycation both lead to increase in hydrodynamic size (dh) of
the protein, although glycation induces a wider size distribution than fructation.
emission peak at 340 nm steadily increases during the first few
days of the reaction until a maximum is reached on day 4, as
shown in Fig. 2(A). The increasing trend of the fluorescence
reverses from the 5th day, which is reflected in Fig. 2(B), showing
the decrease of fluorescence intensity during the 6th, 7th and 8th
days of glycation. In addition to the decrease of intrinsic protein
fluorescence at 340 nm, a new peak appears and grows at 450 nm.
The observed lowering of trp emission (at 340 nm) and concurrent
increase of emission from the glycosylation products (at 450 nm)
can be explained by fluorescence resonance energy transfer
(FRET) from trp to the AGEs.
The upper panel in Fig. 3 describes the time course of trp
fluorescence of the glycated or fructosylated protein. A
comparison of the glucose (dotted line) and the fructose (solid
line) illustrates that the profile has a concavity in case of
fructose, while in case of glucose there is a monotonic increase
in trp fluorescence. The lower panel of Fig. 3 shows the time
profile of the two AGE products. The temporal accumulation of
the two AGE components pentosidine and MDA (ex at 335 nm
and 370 nm respectively) is shown in this figure. The open and
closed symbols respectively represent the effects of fructose and
glucose. For both pentosidine (circles) and MDA (squares) the
temporal accumulation of AGE seems to be higher in case of
fructose.
The trp fluorescence change and AGE formation at various
concentrations of the two sugars indicate a similar trend
(fructose being more effective). It is illustrated in Fig. SM 1 of
the Supplementary Material that:
dðlogAGEÞ=d½f ructose >> dðlogAGEÞ=d½glucose:
where, d/d[sugar] represents the sugar concentration dependent
increase of the AGE product formation.
3.3. Heme release
While Fig. 3 illustrates that glucose is a weaker modifier of
Hb than fructose, the absorption spectra in Fig. 4A show that
236 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
Fig. 2. Hemoglobin (excitation at 280 nm) monitored at different stages of the
fructosylation reaction. (A) Emission spectra on day 0 to day 3 after incubation
in 0.05 M fructose at 37 °C. The arrow indicates that the emission intensity
increases with advancement of fructosylation. (B) Emission spectra on day 6, 7
and 8, showing a decrease in fluorescence near 340 nm (where it was initially
increasing in A). However, a new peak develops around 450 nm representing
formation of AGEs. The isosbestic point at 390 nm may be noted.
glycation (reaction by fructose or glucose) of Hb results in loss
of the heme moiety. Importantly, heme loss with fructation is
much faster than that caused by glucation. Comparison of the
absorbance profiles suggests that while fructose induces com-
plete loss of heme from Hb at as low concentration as 0.1 M,
glucose is not able to do so even at the higher concentration of
0.5 M.
A diminution of the height of the gamma Soret peak and
the overall changes with the length of incubation period (in
0.05 M fructose) is clearly visible from the spectral set shown
in Fig. 4B (A). In Fig. 4B, panel C represents the control
hemoglobin (not incubated with sugars). There are only minor
changes in the spectra (due to conversion to methemoglobin).
Panel B in Fig. 4B represents the temporal profile of changes in
absorbance at 415 nm with progress of fructosylation. Similarly,
panel D represents the kinetics of variations in λmax due to
fructosylation.
A comparison of the heme stain and Coomassie stain of the
native PAGE of glycated hemoglobin (see Fig. SM 2 of the
Supplementary Section) clearly indicates that heme loss is
initiated in the early stage of glycation.
3.4. Alteration in gel mobility
Fig. 5 shows the native and SDS gel migration profiles of
glycated and fructated Hb. The native page (Fig. 5A) shows that
both fructosylated (4th lane from left) and glycosylated bands
(2nd and 3rd lane from left) have migrated longer distances than
the control (extreme left). Additionally, when the effect of
different sugars is compared (3rd and 4th lanes, equimolar
concentrations of glucose and fructose), it is observed that
fructosylated Hb shows higher migration than the glycosylated
one. This might be a result of a shift in tetramer–dimer
equilibrium towards the dimer (as observed in case of glucose-
induced modification) or due to digestion of the protein itself to
produce smaller fragments of different molecular weights (as
found for fructose-induced modification). Two other effects
notable in the SDS PAGE are: (i) presence of a dimeric
population (Fig. 5B) occurring for lower extent of glycation and
(ii) increased fragmentation with higher extent of glycation
(Fig. 5C).
SDS-PAGE analysis (Fig. 5B) of aliquots from the reaction
mixture of Hb in 0.5 M glucose (incubated for 30 days at 27 °C,
shown in lane 3 in Fig. 5A) shows that glycated Hb is composed
of two distinct bands (right lane) with respect to control (middle
lane). Comparison with the marker (left lane) shows that the two
bands correspond to the monomeric (14.4 kDa) and the
crosslinked dimeric (35 kDa) species. Even in presence of
SDS the crosslinking is not broken. On the other hand, the
control hemoglobin results in a single dominant monomeric
band. Fructosylated Hb prepared by incubating Hb in high
fructose concentration (0.5 M) for 30 days at 27 °C produces a
smear (data not shown) when monitored by SDS-PAGE
(implying protein degradation at advanced stages of glycation).
Fig. 3. Changes in intrinsic fluorescence of hemoglobin in presence of 0.05 M
glucose (dotted line) and fructose (solid line), respectively at 37 °C. The upper
panel shows the trp fluorescence emission. The lower panel represents the time
course of two AGE products (pentosidine, excitation 335 nm and MDA,
excitation 370 nm) shown in darker and lighter shades.
 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242 237

Fig. 4. (A) Absorbance spectra in the Soret region of hemoglobin incubated in different concentrations of glucose (left) and fructose (right) for 30 days at 27 °C. In the
right panel, black blue, red and green represent 0.1 M, 0.2 M, 0.3 M and 0.4 M fructose, respectively whereas in the left panel black, blue, light blue, red and green
represent 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5 M glucose, respectively. (B) Kinetics of heme loss during fructosylation. (A) Absorbance spectra of Hb from day 0 to day
6 during incubation with 0.05 M fructose at 37 °C. (B) Kinetics of change at 415 nm (Soret peak) during the modification reaction by fructose. Error bars in this plot
were evaluated on the basis of three independent measurements. (C) Spectra of Hb in absence of fructose (control). (D) Time dependent shift of wavelength of the Soret
peak (λmax) during fructosylation.

Time dependent SDS-PAGE analysis of fructosylated Hb
(Hb in 0.05 M fructose incubated at 37 °C) shown in Fig.
5C also proves that damage induced by fructose is greater
than that induced by glucose. The extreme left lane shows
the marker, while the other lanes illustrate formation of
aggregated cross-linked populations (starting from 0th day to
9th day) due to fructosylation reaction. The cross-linked
dimeric band appears and fades with time and a smeared
zone emerges. Comparison of the time scales in Fig. 5B and
C makes it evident that the kinetics of structural change
induced by glucose is much slower than that induced by
fructose.
3.5. Lowering of alpha helix and elevation of beta sheet and
random coil content
Fig. 6A shows the far-UV CD spectra of hemoglobin
between 190 and 250 nm as a function of time after its
incubation with fructose (0th–7th day), from which it is evident
that fructosylation leads to a systematic alteration in secondary
structure.
Analysis of the CD spectra using the program CONTIN
available at the website DICHROWEB reveals that with
progress of the reaction there is a decrease in the α-helix
content and a compensatory rise in the random coil and β-sheet
238 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242

Fig. 5. (A) Native gel pattern showing changes in mobility of Hb as a result of modification by sugars with respect to the control. The lanes from left represents control
Hb, Hb modified by 0.4 M (2nd) and 0.5 M (3rd) glucose and 0.5 M (4th) fructose, respectively and the temperature of incubation was 27 °C. (B) and (C) SDS-PAGE
pattern of Hb modified by reaction with the sugars. In panel (B), an aliquot from the reaction mixture of Hb in 0.5 M glucose (third from left) was withdrawn after
30 days kept at and analyzed with respect to control Hb (middle lane). Panel (C) shows time dependent SDS-PAGE profile of fructosylated Hb, with samples from the
reaction mixture of Hb and 0.05 M fructose (at 37 °C) being withdrawn for analysis on each day from day 0 to day 9. The extreme left lanes in both show the migration
of markers.

components (Fig. 7). The actual fits of the measured spectra
with the sum of the standard spectra (as provided by CONTIN)
are shown graphically in the Supplementary Materials Section.
The percentages of different types of secondary structure
present in the spectra, emerging from the fits, are compiled in
Table SM1. The experiment was repeated for a slightly higher
incubation temperature of 40 °C. The results of fitting the
second set of CD spectra with the standards using the program
CONTIN are shown in Table SM2, while the actual spectra are
plotted in Fig. SM3(a) (Supplementary Materials Section).
Error bars of the fitting coefficients shown in Fig. SM3(b)
were obtained as the variance of the coefficients given by the
‘closest matching solution’ and those given by the ‘average of
all matching solutions’ by the fitting program CONTIN. It is
observed that uncertainties in the values of two of the
coefficients (those for the beta sheet and the random coil)
increase several-fold for spectra taken after 5 days and later.
However, this does not alter the main result emerging from the
fits, namely, that the fraction of helix present decreases
substantially, while that of both sheet and coil increase, with
increasing number of days of incubation.
CD spectra were also collected in the near-UV and visible
region, which included the Soret region (400–450 nm) (Fig.
6B). They indicate that glycation induces loosening or
detachment of the heme moiety from hemoglobin, caused
possibly by accompanying alterations in the tertiary structure
(250–500 nm). The time dependence of the change of CD
signal in the Soret region matches well the previously observed
kinetics of change in absorbance spectra (Fig. 4B(A)), which
show that Hb gradually loses the heme moiety during
fructosylation after conversion to the met form from the oxy
form [22].
3.6. Protein dependence of fructosylation
To find out the mechanism of AGE formation, a com-
parative study of fructosylation in the three proteins Hb, HSA
(Human Serum Albumin) and lysozyme was carried out. The
results show different extents of fructosylation for the three
proteins (Fig. 8A). The relative amount of AGE formed during
the reaction with fructose was the least for lysozyme. The time
dependence plot of the trp fluorescence intensity for the three
proteins (Fig. 8B) shows that the variation is most prominent
in case of Hb (black curve), for which the rise and fall in
fluorescence represent changes in protein structure. But both
the non-heme proteins, HSA and lysozyme, appear to be less
prone to fructosylation-induced damages than Hb within the
measured period (0th–6th day). However, SDS-PAGE analysis
of progressively older reaction mixtures of fructose with
lysozyme and HSA indicated that fructosylation-induced
damage on day 6 was higher for lysozyme than for HSA (see
L6 and A6 in Fig. 8C).
It has been reported that prolonged glycation leads to
formation of cross-linked beta structure [1]. A comparative
study of different proteins (Fig. 8D) however indicates that in a
relatively short period the beta structure formation is observed
 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
Fig. 6. (A) Far-UV CD spectra showing changes in secondary structure during
the progress of the fructation reaction of hemoglobin incubated in 0.05 M
fructose at 37 °C on different days. (B) CD spectra of hemoglobin incubated in
0.5 M fructose at 37 °C in the near-UV and visible region recorded on different
days during the reaction. A transition from the oxy- (day 0) to met- (day 2) form
of Hb during the fructation reaction causes a spectral shift of the peak in the
Soret region (415 nm), which is followed by decrease in intensity without further
peak shift.
only in case of hemoglobin after an initial small increase in
alpha helix content. Such initial small rise in alpha helix is
observed in each protein. Thus, CD spectra of samples of Hb,
HSA and lysozyme, subjected to fructosylation for a small
duration, are presented in Fig. 8D (detailed in the legend of
Fig. 8D). The CD spectra of hemoglobin are shown on 0th day,
2nd day and 6th day. It is evident that the most appreciable beta
structure formation occurs only in case of Hb. The secondary
structure alteration observed in Hb (beta sheet formation at the
expense of alpha helix) is not observed in either lysozyme or
HSA within this period of fructosylation. Similarly, the
effective rate and extent of AGE product formation is more
in Hb as compared to lysozyme or HSA (Fig. 8A). Of the three
proteins (Hb, HSA and lysozyme), lysozyme contains the least
number of lys or arg (per molecule) residues, which are taken
to be the reaction sites for the sugars: 7 lys and 10 arg for
lysozyme (PDB ID 135L), 42 lys and 14 arg for Hb (PDB ID
1HHO and 4HHB) and 59 lys and 23 arg for HSA (PDB ID
239
1AO6). As the extent (or kinetics) of glycation does not
depend on the number of glycation prone amino acid residues
alone, it was decided to check whether accessibility of such
residues differ in these three proteins. To that end, the
accessible surface area (ASA) of the lysine residues was eva-
luated using the online program ASA-View available at the
website http://www.netasa.org/asaview/ [23]. Using this pro-
cedure it was found that both A and B chains of 1HHO contain
more exposed lysine residues than either HSA (1AO6) or
lysozyme (135L). Fig. SM4 shows spiral plots generated by
ASA-View, which represent this analysis graphically. This
analysis, therefore, suggests that the accessible surface area of
the residues is a key factor responsible for the variability of
fructosylation rates of the proteins.
4. Discussion
The problem of simulating the glycation process in vivo is
that in the cell the glucose concentration is of the order of 5 mM,
which is ten times lower than the concentration used in this
work to study glycation (50 mM). However, it should also be
noted that the hemoglobin concentration used in this study is
1 mg/ml, while it is about 150 mg/ml in normal healthy red
cells. Using a law of mass action logic, in a reaction
A½sugar þ B½proteinYC
the rate of reaction depends on both [A] and [B]. If [B] is lower
while [A] is higher, the situation will be equivocal as far as the
reaction rate is concerned, as compared to high [B] and low [A],
the latter prevailing in the physiological milieu. Given the rates
are similar, the damage will be higher if the protein
concentration is high, as the cross linking may have a cascading
effect not foreseen in the study with the diluted protein. In other
words, though the sugar concentration used seems higher than
the physiological level, a chemically equivalent condition pre-
vails in this study of structural changes. One factor that can
Fig. 7. Fractions of different secondary structure components present in the CD
spectra of Fig. 6B at different stages of the glycation reaction. Analysis of the
CD spectra was performed using the program CONTIN. Qualitatively similar
results were obtained from an analysis using the program K2D.
240 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
attenuate the glycation (and consequently AGE formation) is a
shift of the tetramer ⇔ dimer equilibrium towards tetramers.
The protein (Hb) concentration used for our study shifts this
equilibrium towards dimers and hence there is an increase in
sites for glycation and the rate of glycation is enhanced. Our
preliminary observation confirms this. Any mutation that shifts
the equilibrium towards dimers is thus likely to enhance the
glycation induced damage.
The present work details the complex set of structural
transitions associated with protein glycation. Two structural
features that have been highlighted are: (a) changes in globular
size as revealed from extension of hydrodynamic size; (b)
lowering of alpha helix and elevation of beta sheet and random
coil elements in a compensatory way. While the formation of
beta structure in response to glycation has been reported [1] in
other proteins, this to our knowledge is a first report describing
the compensatory loss of alpha helix with the corresponding
gain of beta secondary structure. A minor temperature shift
(from 37 to 40 °C) does not change the qualitative feature of this
result (helix to sheet conversion). However, there is an
enhancement of the extent to which the conversion occurs at
slightly elevated temperature (see Fig. SM3). Moreover, at this
elevated temperature the small initial rise in helicity prior to beta
structure formation is absent and there is a more prominent
transition to beta structure.
The changes that can explain the glycation induced protein
damage can be traced back to protein cross-linking induced
by glycation at specific sites (say, lysines). The evidence for
such cross-linking is shown by the presence of an additional
SDS resistant dimeric band in the SDS PAGE of glycated
hemoglobin (Fig. 5B). Such covalent cross-linking, in turn, may
induce stress in the backbone finally triggering the overall
structural change. Also, the observed enlargement of hydro-
dynamic size might be explained in terms of higher proportion
of the beta structure, a precursor of protein aggregation. We
further suggest that, in the initial phase with advancement of
Maillard reaction [24] active participation of the free -NH2
group and fructose takes place to form a Schiff's base linkage,
leading to increase in protein fluorescence with no peak at
340 nm. But during the later phase of the reaction a gradual
decrease in trp fluorescence with systematic increase in the peak
at 450 nm is observed. This peak at 450 nm signals formation of
the AGEs and the rise in its intensity reveals their accumulation.
The fact that the absorption peaks of these two glycation
products exhibit strong overlap with the emission peak of trp
(around 335–350 nm, see Fig. 3) it implies existence of an
energy transfer process. This leads to quenching of trp fluo-
rescence at the expense of AGE product fluorescence. One
additionally finds emergence of an isosbestic point in the
Fig. 8. (A) Comparison of the extent of AGE formation in Hb, HSA and
lysozyme during incubation in 0.05 M fructose for 6 days at 37 °C. AGE
formation was estimated by removing an aliquot from the reaction mixture
every 24 h and recording its fluorescence emission intensity at 450 nm, using
excitation at 350 nm. Results are mean ± S.D. of three independent
experiments. (B) Changes in fluorescence emission profile of HSA, lysozyme
and hemoglobin with progress of fructosylation reaction. Data were recorded
for 6 consecutive days of fructosylation reaction and was repeated for 3
measurements. The black curve represents the Hb trp fluorescence pattern as
found earlier (Fig. 3) while the other two proteins are represented by blue
(lysozyme) and green (HSA) curves. (C) SDS-PAGE analysis of HSA and
lysozyme during the fructose modification reaction. (A0, A3, A6) and (L0,
L3, L6) represent samples withdrawn on 0th, 3rd and 6th day from incubation
mixtures of HSA and lysozyme, respectively, with 0.05 M fructose. The
extreme left lane represents the marker. (D) Explains the secondary structure
transition in fructation reaction for Hb, HSA and lysozyme. The CD spectra
in the 190–250 nm region shows a comparison of the spectral data measured
on 2nd and 6th day for hemoglobin and on 6th day for both HSA and
lysozyme including their control proteins.
 R. GhoshMoulick et al. / Biochimica et Biophysica Acta 1774 (2007) 233–242
spectra, indicating a conversion of early glycation adduct to late
stage glycation product. The progress of the reaction on the
other hand also induces a conformational switchover in
hemoglobin, which destroys the heme pocket as well as
weakens the heme-peptide bond. The Soret peak initially shifts
towards the met-Hb form (peak at 406 nm). Met-Hb formation
further facilitates loss of heme from Hb during fructosylation.
There are certain pertinent questions that may deserve mention
at this stage. The heme loss associated with glycation of
hemoglobin may be important in dealing with diabetic
complications co-occurring with anemia [7,8].
While the structural alteration reported above is found to
be similar among different proteins, the extent of glycation-
induced change may vary. It is shown that the extent does
not directly depend on the number of glycation prone
residues alone, but on the solvent-accessible surface area of
such residues. Although Hb contains fewer lys residues
compared to albumin, the amount of AGEs and the glycation
induced damage in the former are much higher than the
latter. The fact that beta structure is formed in case of
albumin subjected to prolonged glycation has already been
reported [1]. One can offer some arguments explaining this
faster glycation (and glycation induced structural change) in
hemoglobin.
As Hb is a better target for glycation than albumin, it is
expected that aggregation of glycated Hb will have more
adverse effect on the system than aggregation of albumin. The
predominant presence of albumin in the serum, however,
amplifies its risk and similarly the splenic degradation of red
cells and the proteins therein removes the damage from the
system.
Similarly, the emergence of beta structure with glycation
suggests possible therapeutic approaches based on negative re-
gulation of beta structure formation. According to this scenario,
a putative glycation inhibitor might function by arresting the
alpha structure or minimizing beta structure formation.
5. Conclusion
Glycation induces a set of complex structural change in
hemoglobin. The primary damaging effect is onset of a
compensatory secondary structure alteration, namely, loss of
alpha helix and increase in beta sheet structure. The associated
effects include heme loss and protein aggregation and an
anomalous increase of trp fluorescence followed by its decrease.
While the increase in fluorescence may be due to protein
unfolding, the decrease may be a result of FRET between trp
and an AGE product (pentosidine) which has excitation maxima
near the emission maxima of trp. As the formation of AGEs and
onset of beta structure formation occur almost simultaneously, it
may be conjectured that beta sheet formation is a result of
accumulation of AGEs induced protein cross-linking.
Furthermore, between the two sugars studied, fructose
showed faster glycation kinetics than glucose. Thus patients
with any defect in fructose metabolism pathways are predicted
to have more glycation induced protein damage (or AGEs
formation). Lastly, the accessibility of glycation sites rather
241
than their total number serve as a rate determining factor for
glycation, and this may explain why hemoglobin is more
prone to glycation induced damage than proteins containing
much higher number of lysines. By the same arguments
patients with lower hemoglobin concentration (with the
tetramer–dimer equilibrium shifted towards the more exposed
dimer) are more likely to suffer from glycation induced
damage. The heme loss associated with glycation, and this
increased accessibility to glycation at lower hemoglobin
concentrations, indicate existence of an intricate relationship
between diabetes and anemia.
Acknowledgements
We thank the Department of Science and Technology of the
Government of India for financial support through grant no. SP/
SO/D15. We also thank Ms. Sinu Jasrapuria, Mr. Tapan Sarkar
and Ms. Suryyani Deb for assistance. We sincerely thank Prof.
Dr. Prabir Lahiri, Calcutta Medical College, Prof. J. Jean-Luc
Coll, Recherche sur le Cancer du Poumon, France and Prof.
Andreas Offenhaeusser, Institute of Thin Films and Interfaces,
Germany, for helpful discussions and encouragement.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bbapap.2006.11.018.
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