Abstract-- The conformational
stability of bovine serum albumin (BSA) against urea denaturation was
investigated in aqueous solutions both in the absence and presence of buffers.
Various buffers differing in polar and nonpolar characters such as sodium
phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES
and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this
study. Urea-induced structural changes were analyzed using different spectroscopic
techniques, i.e., intrinsic fluorescence, ANS fluorescence and
UV-difference spectroscopy. The presence of different buffers in the
incubation medium offered different degrees of resistance to the protein
against urea-induced structural changes. A similar trend of buffer-induced
structural resistance was noticed with three different techniques. The
stabilizing effect of these buffers followed the order: MOPS > HEPES >
sodium phosphate > Tris-HCl > pure water. The highest stability of BSA observed
in MOPS, and HEPES buffers can be attributed to the presence of morpholine and
piperazine rings in their structures, respectively. These groups might have
produced a hydrophobic environment around the protein surface, stabilizing
protein conformation against urea denaturation.
Keywords-- bovine serum albumin,
HEPES, MOPS, protein stability, urea denaturation.
I. INTRODUCTION
The subject of protein stability has been
widely studied to get insights about retaining protein’s biological activity and
functionality in any required environment. Each protein has its own stability behavior
depending on its unique three-dimensional structure, maintained and supported
by several forces. These forces include hydrogen bonds, van der Waals
interactions, ionic interactions, disulfide bonds and above all, hydrophobic
interactions as the leading force (Dill, 1990). The native state of a protein
has been found marginally stable than its denatured state. In other words, it
can be readily denatured by the alterations in the environment, i.e.,
changes in the salt concentration, pH, temperature or presence of chaotropic agents
such as urea or guanidine hydrochloride that can disrupt noncovalent forces
required to maintain the native structure (Majumdar et al., 2013, Gupta et
al., 2015, Kandandapani et al., 2016, Pannuru et al., 2018, Brudar
and Hribar-Lee, 2019). A disruption in the protein’s three-dimensional
structure subsequently leads to a loss in the protein’s function. Hence, it is crucial
to find ways to retain their native structure under different environments so
that their functions are maintained. This is especially important in
pharmaceutical research to produce useful therapeutic protein products.
Serum albumin is known as the primary carrier for
various molecules in the bloodstream. In the present study, bovine serum
albumin (BSA) was selected as a model protein due to its structural stability,
easy availability, and lower cost (Peters, 1985, Carter and Ho, 1994). BSA denaturation
by urea has been shown to occur in a two-step, three-state transition with the
start-point, the mid-point and the end-point, at around 3 M, 5 M and 8 M urea,
respectively, when studied by intrinsic fluorescence measurements (Tayyab et
al., 2000). Buffers play an essential role in the functioning of a protein,
and these can mimic the conditions of the body fluids. Buffers help regulate the
intracellular and extracellular pH of the body fluids (Reddy et al.,
2015). Phosphate and bicarbonate buffers constitute two crucial buffer systems
in the human body. The phosphate buffer is useful in the cytoplasm of all cells
(Thomas and O'Shea, 2005). The role of the bicarbonate buffer system is to
maintain the blood pH of mammals. It manages the acid/base imbalances and
effectively controls excess carbon dioxide released as a by-product of cellular
respiration (Fitzharris and Baltz, 2006, Fitzharris et al., 2007,
Zander-Fox et al., 2010). Proteins’ native three-dimensional structures
are maintained by these buffers both inside the cells and outside compartments.
Pannuru et al. (2018) and Pavani et al. (2021a, 2021b) have shown
the stabilizing effect of biological buffers, including Tris and sodium
phosphate buffers, on the thermal stability of proteins and enzymes through
protein-buffer interactions. Several studies have shown the influence of
buffers on protein denaturation due to changes in ionic strength or pH (Gupta et
al., 2015, Kandandapani et al., 2016, Salis and Monduzzi, 2016).
However, the intrinsic influence of buffer components on the protein structure
and stability remains unclear. This is important to understand the factors that
enhance protein stability. In this context, we studied the effects of buffers
on the conformational stability of BSA against urea denaturation using
fluorescence and ultraviolet (UV)-difference spectroscopy.
II. MATERIALS AND METHODS
A.
Materials
Fatty acid-free bovine serum albumin (BSA) (Lot No. 080M7405V),
1-anilinonaphthalene-8-sulfonic acid (ANS) (Lot No. 104K2510) and urea (BioXtra,
pH 7.5-9.5, 20°C, 5M in H2O) were purchased from
Sigma Aldrich Inc. (USA). MOPS, sodium salt and HEPES, sodium salt were
obtained from Calbiochem® (Germany), while Tris was obtained from
AMRESCO® (USA). Sodium dihydrogen phosphate and disodium hydrogen
phosphate were the products of SYSTERM® (Malaysia). Deionized
distilled water was used throughout this study. All the experiments were
performed at room temperature (~25°C) unless otherwise stated. Analytical
grade samples of other chemicals were used.
B.
Analytical procedures
The stock solutions of BSA and ANS were prepared
in either deionized water or respective buffers at pH 7.0 (0.2 M Tris-HCl, 0.2
M sodium phosphate, 0.2 M HEPES and 0.2 M MOPS buffers) by adding a fixed
amount of their powders in a fixed volume of the solvent at room temperature
without agitation. The protein solution was stored at 4oC and used
within a week, while the ANS solution was prepared fresh and used on the same
day. Their concentrations were determined
spectrophotometrically with a Shimadzu double-beam spectrophotometer, model
UV-2450, using quartz cuvettes of 1 cm path length. The molar extinction
coefficient values of 43,824 M-1 cm-1 at 279 nm for BSA (Peters,
1975) and 5000 M-1 cm-1 at 350 nm for ANS (Mulqueen and
Kronman, 1982) were applied for concentration measurements. The urea (8 M)
stock solution was also prepared in either deionized water or various buffers,
and its concentration was determined by following the procedure described by
Pace and Scholtz (1997).
C.
Fluorescence spectroscopy
Fluorescence measurements were carried out on a Hitachi fluorescence
spectrophotometer, model F-2500, equipped with a data recorder. The excitation
and emission slits were fixed at 10 nm each. The intrinsic fluorescence spectra
of the protein solutions in pure water or various buffers were recorded at the
wavelength range of 310-400 nm, using an excitation wavelength (lex) of 295 nm and protein concentration of 2 µM in a quartz cuvette of
1 cm path length. The fluorescence spectra were also corrected using
appropriate blanks, prepared in the same way but without protein.
ANS fluorescence spectra of the samples containing ANS
(140 µM) and the protein (2 µM) were recorded at the wavelength range, 400-600 nm, using lex of 380 nm. The molar ratio between ANS and protein was fixed at
70:1 throughout the measurements, and the fluorescence spectra were corrected
by subtracting the fluorescence contribution of the appropriate blanks.
D.
UV difference spectroscopy
The UV absorption spectra of BSA (20 µM) in different buffers were
measured with the Shimadzu double-beam spectrophotometer, model UV-2450 at 25°C at the
wavelength range, 250-300 nm. The absorbance values of the protein (in buffer) were
subtracted from the protein’s absorbance values (in urea) for each of the urea
concentrations at each wavelength to obtain the UV- difference spectra. These
values were then transformed into differential extinction coefficient (De) by dividing the absorbance
difference of each sample by the molar concentration of BSA and were plotted
against wavelength.
E. Denaturation experiments
Different volumes of the buffer solution were added to the 0.5 mL
BSA stock solution, taken in different tubes. After 5 min incubation, the
required volume of the urea stock solution was added to these tubes to obtain
the desired urea concentration.
For the ANS experiment, 0.1 mL of the BSA solution was
mixed with 0.1 mL ANS stock solution in the appropriate buffer volume. The
mixture was incubated for 10 min before adding the appropriate urea stock
solution to achieve the desired urea concentration.
The blanks were also prepared in the same way as
described above but without protein.
The reaction mixtures were incubated overnight at room
temperature before recording the fluorescence/absorption spectra.
III. RESULTS AND DISCUSSION
Intrinsic fluorescence, ANS fluorescence, and UV difference spectroscopic
techniques were used to study the effects of various buffers on the structural
stability of bovine serum albumin in the presence of urea.
A.
Intrinsic fluorescence spectra
Urea
denaturation has been employed to study the conformational changes in a protein
and gain insight into its unfolding process. Molecular dynamics simulations of
urea denaturation suggest that several mechanisms are involved in this process
in which urea competes with backbone hydrogen bonds, influences solvation of
the hydrophobic core and affects the water structure, thus alters the
hydrophobic effect (Bennion and Daggett, 2003). Such an effect on the protein's
hydrophobic core may disrupt the protein conformation (Lim et al., 2009,
England and Haran, 2011). As the urea concentration rises, the protein (BSA)
molecule starts to unfold (~3.0 M urea) and becomes completely unfolded at 8.0 M urea
concentration (Tayyab et al., 2000). The change in the protein
conformation due to the addition of urea can be investigated by intrinsic
fluorescence measurements, which monitor microenvironmental alteration around
the protein fluorophores. The responsive fluorophores in the protein are the
aromatic amino acid residues, namely, tryptophan (Trp), tyrosine (Tyr) and
phenylalanine (Phe). However, upon excitation at 295 nm, the fluorescence
spectrum indicates the microenvironment around the Trp residues in the protein
molecule (Lakowicz, 2013, Tayyab et al., 2019; Musa et al.,
2020).
Figure 1 (A-E) shows the urea-induced structural changes in BSA in the absence
and presence of different buffers, as monitored by fluorescence spectral
measurements, upon excitation at 295 nm. BSA in pure water produced a
fluorescence spectrum at the wavelength range of 310-400 nm with an emission
maximum at 340 nm (Fig. 1A) due to tryptophan residues (Trp-134 and Trp-213).
These residues are located at the surface of subdomain IB and within the
hydrophobic binding pocket of subdomain IIA, respectively (Tan et al.,
2013). As can be seen from Fig. 1A, the fluorescence spectra showed progressive
quenching of the protein fluorescence and blue shift in the emission maximum in
the presence of 4-6 M urea. The value of the relative fluorescence intensity at 340 nm
(RFI340 nm) quenched from 100 (in pure water) to 78.7 and 67.0,
corresponding to a percentage quenching of 21.3 % and 33 % at 4.0 M and 5.0 M
urea, respectively (Fig. 1A, Table 1). A slight blue shift of 3 nm and 4 nm in
the emission maximum of BSA at 4.0 M and 5.0 M urea was also observed. The
occurrence of the blue shift indicated that the microenvironment around Trp residues
of the protein became more nonpolar (Brodersen et al., 1977,
Lakowicz, 2013). However, the quenching reflected a
significant conformational change in the native protein. At 6.0 M urea
concentration, a large decrease in the RFI340 nm value (50.8 % quenching)
suggested a greater degree of protein denaturation. Qualitatively, a similar
pattern of quenching in the fluorescence spectra of BSA was seen in all four
buffers. However, perceptible differences in the RFI340 nm values of
BSA at 4.0 M, 5.0 M and 6.0 M urea were realized in various buffers, as shown
in Fig. 1 and Table 1. A significant reduction in the percentage quenching of
RFI340nm value of BSA was observed for Tris-HCl buffer and sodium
phosphate buffer. The percentage quenching in the RFI340 nm was 13
%, 22.9 % and 45.4 % in Tris-HCl buffer, which were reduced to 8.3 %, 18.1 %
and 39.2 % in sodium phosphate buffer at 4.0 M, 5.0, and 6.0 M urea,
respectively (Fig. 1, Table 1). These results indicated that sodium phosphate
buffer offered relatively better resistance in the urea-induced structural
change than the one offered by Tris-HCl buffer.
Interestingly, HEPES and MOPS buffers showed a
somewhat similar quenching pattern in the RFI340 nm within the 4.0
to 6.0 M urea concentration range. At 4.0 M urea, BSA in the HEPES buffer
showed a 5.9 % quenching in the RFI340 nm than the one showed by
native protein, while a 2.9 % quenching was observed in the MOPS buffer.
Similarly, at 6.0 M urea, the percentage quenching was 28.9 % and 28.2 % in
HEPES and MOPS buffers, respectively. A comparison of the values of the
urea-induced quenching in the RFI340 nm, obtained in different
buffers, suggested that BSA showed a lesser extent of quenching in MOPS and
HEPES buffers compared to that observed in Tris-HCl and sodium phosphate
buffers. These results indicated relatively lesser urea-induced structural
changes in BSA in the presence of MOPS and HEPES buffers. In other words, MOPS
and HEPES buffers offered better protection to BSA against urea denaturation.
Hence, BSA seems to be relatively stable against urea
denaturation in MOPS and HEPES buffers, followed by sodium phosphate and
Tris-HCl buffers, while it is least stable in pure water. Thus, it can be said
that the presence of a buffer offers greater stability to the protein compared
to its absence. Furthermore, different buffer composition produces different
influences towards BSA structural stability due to differences in their
structures. This seems reasonable given the hydrophobic environment produced by
the piperazine ring of HEPES and the morpholine ring of MOPS around surface
residues of BSA.
B.
ANS fluorescence spectra
Due to the presence of hydrophobic
clusters in the proteins, a well-known hydrophobic dye,
8-anilino-1-naphthalenesulfonic acid (ANS), is widely used to explore the conformational changes in proteins
upon folding / unfolding (Brudar and Hribar-Lee, 2019, Ptitsyn, 1995,
Hawe et al., 2008). ANS-protein
complex is formed through noncovalent hydrophobic interactions. The higher
emission intensity of ANS is usually shown when bound to the molten globule
state with respect to the native or denatured states of a protein (Ptitsyn,
1995, Muzammil et al., 1999; Hawe et al., 2008, Abd Halim et
al., 2014). Loss of ANS fluorescence
with denatured protein indicates either disruption of ANS binding sites upon
unfolding (Ptitsyn, 1995) or displacement of bound ANS molecules at higher
denaturant concentration (Kumar et al., 1996). Urea-induced changes in
the protein (BSA) conformation in the absence and presence of four different
buffers, as monitored by ANS fluorescence intensity upon excitation at 380 nm,
are depicted in Fig. 2. A marked quenching in the ANS fluorescence spectra of
BSA was observed upon increasing urea concentration from 4.0 M to 6.0 M in both
pure water and different buffers. However, the quenching in the ANS
fluorescence intensity showed different magnitudes at the highest urea
concentration (6.0 M). Such differences in the magnitude of quenching in the
ANS fluorescence signal of BSA in the absence and presence of different urea
concentrations can be viewed from Table 1. The relative fluorescence intensity
at 472 nm (RFI472 nm) of BSA in pure water was decreased up to 40.1
(59.9 % quenching) at 6.0 M urea (Fig. 2, Table 1). These results indicated
protein denaturation due to disruption of the protein’s hydrophobic clusters in
the presence of urea. Since BSA in the pure water at 6.0 M urea yielded the
lowest RFI472 nm value, maximum disruption of the hydrophobic
clusters can be considered at this urea concentration.
In the Tris-HCl buffer, the RFI472 nm value was reduced
to 46.1 at 6 M urea, which was higher than the RFI472 nm value
obtained in pure water (40.1) but lower than that achieved in sodium phosphate
buffer (54.0). To make it clear, increasing the urea concentration from 4.0 M
to 6.0 M, the percentage quenching in the RFI472 nm was 18% and 53.9
%, respectively, in Tris-HCl buffer than 10.8% and 46 %, respectively, in
sodium phosphate buffer (Fig. 2, Table 1). These results indicated more
disruption of hydrophobic clusters in Tris-HCl buffer than in sodium phosphate
buffer at the same urea concentration. Following the intrinsic fluorescence results,
the lowest quenching in the RFI472 nm value was also observed in
HEPES and MOPS buffers with this probe (Fig. 2, Table 1).
In HEPES and MOPS buffers, the percentage quenching in RFI472
nm between native and 6.0 M urea was 39.3 % and 37.8%, respectively
(Table 1). Since a relatively lesser quenching in the RFI value was noticed in
buffers compared to that observed in pure water, it can be said that there was
some degree of protection of BSA structure against urea denaturation in the
presence of buffers, being significantly higher in HEPES and MOPS buffers.
Quantitatively, a similar pattern of buffers’ influence on BSA structural
stability was noticed in the order of pure water< Tris-HCl < sodium
phosphate < HEPES < MOPS.
Taken together, both ANS fluorescence and intrinsic fluorescence
results suggested that BSA was relatively most stable in HEPES and MOPS buffers
against urea denaturation. The lesser quenching in the ANS fluorescence
intensity of BSA in 6.0 M urea in these two buffers compared to Tris-HCl or
sodium phosphate buffers indicated significant protection of hydrophobic
clusters. Although these results were similar to intrinsic fluorescence
results, showing a similar trend of urea-induced protein denaturation in these
buffers, a higher degree of quenching in the ANS fluorescence intensity
suggested greater sensitivity of ANS fluorescence compared to intrinsic
fluorescence signal that indicated the changes in the microenvironment around
Trp residues in BSA. Since there are only two Trp residues in BSA, the quenching
in the RFI value may not be as prominent as found with hydrophobic clusters.
C.
UV difference spectra
Figure 3
shows the influence of different buffers on the urea-induced structural changes
in BSA, as monitored by the UV difference spectral signal at 287 nm (Δe287 nm). The difference spectra of BSA in different urea concentrations
were characterized by a negative peak at 287 nm and a trough at 280 nm, which
suggested a change in the microenvironment around Tyr residues of BSA in the
presence of urea (Sogami and Ogura, 1973). The negative
signal
at 287 nm became more pronounced with the increase in the urea concentration
(Fig. 3). The strongest spectral signal was observed in the absence of buffer
(pure water) with the Δe287 nm value of – 4.85 in 6.0 M urea
(Table 1).
In various buffers systems, the spectral signal was reduced to a
significant extent. For example, the Δe287 nm
value of BSA at 6.0 M urea was – 4.25 in Tris-HCl buffer, – 4.10 in sodium
phosphate buffer, – 3.70 in HEPES buffer and – 3.25 in MOPS buffer (Table 1).
Out of the four buffers used, HEPES and MOPS buffers showed much-reduced
signals, reflecting the stabilizing effect of these buffers towards the protein
structure. Since the Δe287 nm value showed a lesser change in
HEPES and MOPS buffers at 4.0-6.0 M urea, it suggested retention of significant native structure
in the protein.
The UV difference spectral results also showed a similar sequence of
buffers in stabilizing protein conformation, as realized with intrinsic
fluorescence and ANS fluorescence techniques. HEPES and MOPS buffers contain
piperazine ring and morpholine ring, respectively, in their structures, which
provide a hydrophobic environment around surface residues of BSA, thus
stabilizing the native structure and protecting it against urea denaturation.
IV. CONCLUSIONS
In summary, the structural stability of BSA against urea
denaturation was evidenced from the magnitude of three spectral signals:
intrinsic fluorescence, ANS fluorescence, and UV-difference spectral signal. Whereas
protein fluorescence and absorbance signals probed changes in the
microenvironment around Trp and Tyr residues, disruption of the hydrophobic
clusters was analyzed by the ANS fluorescence in the presence of urea.
Different degrees of structural resistance to the protein against urea
denaturation was seen in the presence of different buffers. In comparison, BSA
in HEPES and MOPS buffers showed greater stability than in sodium phosphate and
Tris-HCl buffers. The greater hydrophobic character of HEPES and MOPS buffers
seems to stabilize the BSA structure against urea denaturation. These findings
will help understand the best environment for proteins to retain their
structural stability.
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Received: February 3, 2021
Sent to Subject Editor: March 18, 2021
Accepted: August 4, 2021
Recommended by Subject Editor Sebastian Collins
