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Biochimica et Biophysica Acta 1699 (2004) 191–199

Molten globule-like folding intermediate of asialofetuin at acidic pH

Farah Naseem, Basir Ahmad, Mohd. Tashfeen Ashraf, Rizwan Hasan Khan*

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India

Received 1 August 2003; received in revised form 24 February 2004; accepted 25 February 2004

Available online 19 March 2004

Abstract

In our earlier communication on acid-induced unfolding of bovine serum fetuin (BSF), we showed the existence of a molten globule (MG)-

like state of BSF at pH 1.8. The MG state was characterized by higher content of secondary structure than native and almost complete loss of

tertiary structure and more solvent exposed hydrophobic surface [Biochim. Biophys. Acta 1649 (2003) 164]. In this work we have shown the

presence of an MG-like partially folded intermediate of asialofetuin at around pH 1.8, which is much different from the MG state observed in

BSF in secondary structure contents. The results show that asialofetuin at pH 1.8 retains f 45% secondary structure, as evident from far-UVCD

spectra. The near-UV CD spectra showed almost complete loss of tertiary structure. The intrinsic fluorescence and acrylamide quenching of the

lone tryptophan residue showed that in acid-induced state, it is buried in the interior in a nonpolar environment. The temperature dependence of

far-UV CD signal of asialofetuin at pH 1.8 exhibits a weak cooperative thermal transition. A significant increase in ANS fluorescence showed

extensive solvent exposure of nonpolar cluster. Size exclusion chromatography (SEC) indicates a slight increase in the hydrodynamic size of

acid-induced protein. These results suggest that asialofetuin at pH 1.8 represents the MG-like folding intermediate. Moreover, our results

showed that glycosylation might play a role in stabilization of secondary structure during acid and/or thermal denaturation.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Asialofetuin; MG state; Circular dichroism; pH; Acid-unfolding

1. Introduction

To understand fully the conformational behavior of a

protein, it is necessary to define not only the structure of its

native state but also that of various denatured states. The

molten globule (MG) state is the intermediate between the

native and unfolded states [1] in which the protein molecule

is almost as compact as in the native state [2,3] and has a

loosely packed nonpolar core [4]. MGs have now been

observed for a significant number of proteins, and it is

assumed that most if not all proteins can form such species.

In other words, MGs are general intermediates in protein

1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbapap.2004.02.011

Abbreviations: MG, molten globule; CD, circular dichroism; GnHCl,

guanidinium hydrochloride; ANS, 8-anilinonaphthalene-1-sulfonic acid;

NATA, N-acetyl-L-tryptophanamide; SEC, size exclusion chromatography

* Corresponding author. Tel.: +91-571-272-0388; fax: +91-571-272-

1776.

E-mail addresses: rizwanhkhan@hotmail.com,

rizwanhkhan1@yahoo.com (R.H. Khan).

folding. The development of broad range of techniques has

led to the identification and characterization of stable

intermediates in several proteins [5–7] Recently a number

of equilibrium intermediates have been detected and char-

acterized, like in our earlier communications we have

reported the MG states in proteins like a-chymotrypsino-

gen-A, stem bromelain, glucose-oxidase [8–10] and fetuin

[11]. The high cooperativity and complexity of the protein

folding process makes the characterization of conformation-

al transitions and equilibrium intermediate states hard to

achieve; different methods are employed for the character-

ization of the folding intermediates and for understanding

the different factors leading to the formation and stabiliza-

tion of these intermediates. One of the oldest known

methods of denaturing proteins is by the addition of acids.

Acid-induced unfolding of proteins varies from apparently

fully unfolded to substantial remaining structure [12].

In the present study, we have characterized MG-like

partially folded intermediate of asialofetuin. Asialofetuin

is the desyalylated form of fetuin.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199192

Fetuin is a glycoprotein abundant in calf serum. It is a

protein found predominantly during fetal stage. Experi-

ments with serum from calves of different ages have

shown that the amount of this globulin has its highest

value in newly born calves and decreases with time. It is

associated with the period when the greatest building and

development of the animal takes place. The carbohydrate

portion of fetuin is made up of three branched hetero-

polysaccharide units. These units have been shown to have

a similar monosaccharide composition. The peptide portion

consists of a single chain with six intrachain disulfide

bonds. The carbohydrate chains comprise 24% of the total

molecular weight, Mr 48,000 Da [13]. Equivalent of fetuin

in other mammalian species is alpha 2-HS glycoprotein

(AHSG) [14]. Fetuin is thought to play an important role

in fetal brain development [15,16] and neonatal skeletal

development [17]. It is also important for postnatal bone

growth and remodeling [18] and contributes to insulin

resistance during normal pregnancy and gestational diabe-

tes [17]. It has been shown that fetuin and various

cytokines have a developmentally regulated appearance

and expression in the developing neocortex [15,16]. A

short 19-amino-acid sequence of fetuin shows a degree of

homology to an 18-amino-acid sequence of the transform-

ing growth factors—beta (TGF-beta) type-II receptor, and

in vitro fetuin binds to members of the TGF-beta family of

cytokines. It has been suggested that fetuin is the biolog-

ically significant antagonist of these cytokines [16]. Thus,

fetuin has a very important role in the overall development

of the fetus. Any misfolding in this protein can have

adverse effects on its functioning and its interaction with

cells and other molecules leading to general development

as well as brain development anomalies. Folding studies

on fetuin and knowledge of its different structural aspects

may prove useful in better understanding of its folding

pathway so that attempts can be made to correct the

misfolding.

In our previous studies on fetuin, it has been shown that

MG state exists at pH 1.8, which has substantial amount of

secondary structure and significantly disordered tertiary

structure; this was inferred on the basis of CD spectra taken

in the far as well as near-UV regions and fluorescence

studies. Thermal studies in the far-UV region showed that

the secondary structure is quite stable and is resistant to high

temperatures while thermal spectra in the near-UV region

were indicative of cooperative unfolding at pH 7 and loss of

cooperativity, i.e. non-cooperative unfolding at pH 1.8,

which again suggested the presence of MG state at this

pH. Based on our previous results on the characterization of

MG state of BSF at low pH, we report here in a systematic

way the conformational behavior of asialofetuin in the

acidic region and contribution of the sialic acid in main-

taining the structural integrity of the protein. For this

purpose, fluorescence and CD spectroscopic measurements

were performed depending on pH. The results are discussed

in the context of known properties of BSF.

2. Materials and methods

2.1. Materials

Asialofetuin from fetal calf serum type-I (lot-90K7650)

and GnHCl were purchased from Sigma Chemical Co. (St.

Louis, MO, USA). Acrylamide was purchased from Quali-

gens Fine Chemicals (India). All other reagents and buffer

compounds used were of analytical grade.

2.2. Methods

A stock solution of 10 mg ml� 1 protein was prepared in

distilled water and it was dialysed for 4 h at low temperature

(4 jC) and the respective solutions were made in different

pH buffers. GnHCl solution (6 M) was prepared in 10 mM

sodium phosphate buffer, pH 7.

2.2.1. Protein concentration determination

Protein concentrations were determined spectrophoto-

metrically using an extinction coefficient E2781% = 5.3 [19]

on a Hitachi U-1500 spectrophotometer or alternatively

by the method of Lowry et al. [20]. pH measurements

were carried out on an Elico digital pH meter (model

LI610).

2.2.2. CD measurements

CD measurements were carried out with a Jasco spec-

tropolarimeter, model J-720, equipped with a microcom-

puter. The instrument was calibrated with D-10-camphor-

sulfonic acid. All the CD measurements were carried out at

25 jC with a thermostatically controlled cell holder at-

tached to a Neslab RTE-110 water bath with an accuracy of

F 0.1 jC. Far-UV CD spectra were measured at a protein

concentration of 3.0 AM and near-UV CD spectra were

measured at protein concentration of 30 AM. The path-

lengths were 1 mm and 1 cm, respectively. The results are

expressed as mean residue ellipticity (MRE) in deg cm2

dmol� 1 defined as

MRE ¼ hobs ðmdegÞ=10� n� Cp� l

where hobs is the CD in millidegree; n = 309 is the number

of amino acid residues; l is the pathlength of the cell in cm

and Cp is the molar fraction. The a-helical content of fetuin

was calculated from the MRE value at 222 nm using the

following equation as described by Chen et al. [21].

% a-helix ¼ MRE222 � 2340=30300� 100:

For thermal-transition studies, a water-jacketed 1-mm

pathlength cell was used for far-UV CD and a water-

jacketed 10-mm pathlength cell was used for near-UV CD

attached to the RTE-110 water bath interfaced with a

microcomputer. A protein concentration of 30 AM was used

for near-UV CD measurements and 3 AM was used for far-

UV CD measurements.

Fig. 1. pH dependence of CD spectra of asialofetuin at 222 nm. Protein

concentration was 3 AM. Experiments were carried out at 25 jC in 10 mM

of the following buffers: pH 1–2.8, glycine-HCl buffer; pH 3–5, sodium-

acetate buffer; pH 6–7, sodium-phosphate buffer.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199 193

2.2.3. Fluorescence measurements

Fluorescence measurements were performed on a Shi-

madzu spectrofluorometer, model RF-540. The fluorescence

spectra were measured at a protein concentration of 3 AM.

Solutions were prepared in buffers 20 mM each of different

pH values ranging from pH 0.6 to 10 [pH 1–2.8, glycine-

HCl buffer; pH 3–5, sodium-acetate buffer; pH 6–7,

sodium-phosphate buffer]. Before making measurements

the solutions were incubated overnight at 4 jC. Intrinsicspectra were recorded between 300 and 400 nm with

excitation wavelength of 280 and 295 nm. For ANS

fluorescence in the ANS binding experiments, the excitation

was set at 380 nm and the emission spectra were taken in the

range of 400–600 nm.

2.2.4. Quenching experiments

In the quenching experiments, aliquots of 5 M quencher

stock solution were added to protein solutions (3 AM) to

achieve the desired range of quencher concentration (0.1–

1M). Excitation was set at 295 nm in order to excite

tryptophan residues only and the emission spectrum was

recorded in the range 300–400 nm. The decrease in fluo-

rescence intensity at kmax was analyzed according to the

Stern–Volmer equation [22].

F0=F ¼ 1þ Ksv½Q�

where F0 and F are the fluorescence intensities at an

appropriate wavelength in the absence and presence of

quencher, respectively, Ksv is the Stern–Volmer constant,

and [Q] is concentration of the quencher.

2.2.5. Size exclusion chromatography (SEC)

SEC experiments were performed on a Sephacryl-S 100

HR (60� 0.8) column. The column was pre-equilibrated

with 25 mM sodium phosphate buffer pH 7 and glycine

HCl buffer of pH 1.8. Two milliliters of 5 mg/ml native

and pH-treated protein solution was applied to the column

and eluted at 20 ml/h. The eluted fractions were read at

280 nm.

3. Results and discussion

In our previous report, we have shown that an MG state

exists in bovine serum fetuin (BSF) at low pH [13]. In order

to monitor the role of charged sialic acid residues in

stabilization of native conformation of the protein, we have

performed similar studies on asialofetuin.

As shown in Fig. 1, the conformation of polypeptide

backbone of asialofetuin was monitored by ellipticity

measurements in the far-UV region at 222 nm. The

MRE at 222 nm showed no apparent change between

pH 7 and 4.0, but when pH was decreased below 4.0,

MRE222 decreased markedly to a minimum value at

around pH 2.4. Further, no apparent change was observed

in the pH range 2.4–1.0. Thus, the pH-induced transition

was found to follow a single step two state transition. The

observed pH denaturation curves as monitored by MRE

measurements at 222 nm of asialofetuin and its sialated

form fetuin were markedly different. The results of sec-

ondary structure resolved analysis are presented in Table 3

for fetuin and asialofetuin.

To detect and characterize the tertiary structure of asia-

lofetuin, near-UV CD, intrinsic tryptophan fluorescence

measurements and ANS binding experiments were used.

Fig. 2a and b shows the fluorescence spectra of asialo-

fetuin in the native state at pH 7, acid-unfolded state at pH

1.8 and completely denatured state in the presence of 6 M

GnHCl in the 300–400 nm range after exciting the protein

at 280 and 296 nm, respectively.When protein was excited

at 280 nm (Fig. 2a), a significant increase in fluorescence

intensity of acid-unfolded protein (curve 2) was observed as

compared to native (curve 1) and GnHCl-denatured protein

(curve 3) along with a red shift of approximately 12 nm as

compared to that of native state; this increase in fluores-

cence intensity may be ascribed to the disruption of hydro-

gen bonding between the hydroxyl group of tyrosyl residue

and carboxyl groups of peptide backbone emphasized by the

loss of secondary structure of asialofetuin in acid-induced

state [23,24]. In the presence of 6 M GnHCl, fluorescence

intensity was more and kmax shifted to longer wavelength

from native protein. On the other hand, when asialofetuin

was excited at 296 nm (Fig. 2b), fluorescence intensity of

completely denatured protein (curve 3) was slightly more

than that of the acid-induced state (curve 2) as well as native

state (curve 1) because at 296 nm there was no excitation of

the tyrosine residues, a slight red shift of 4 nm was

observed. Slight red shift of kmax when protein was excited

at 296 nm shows little change in the tryptophanyl environ-

Fig. 2. Fluorescence emission spectra of asialofetuin when the protein was excited at 280 nm (a) and 296 nm (b). Asialofetuin in the native state at pH 7 (—), in

acid-unfolded state at pH 1.8 (- - -), in 10 mM glycine-HCl buffer and in the presence of 6 M GnHCl (. . .). Protein (3 AM) was incubated with 10 mM buffers of

different pH for 3 h. Spectra were recorded in the range 300–400 nm. Experiments were carried out at 25 jC.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199194

ment, which is also evident from Fig. 6 where MRE290 of

acid-unfolded protein is almost similar to that of native

protein. Red shift of kmax in both cases, i.e. when protein

was excited at 280 and 296 nm, is indicative of more polar

environment of the single tryptophan residue, which may be

either due to exposure of tryptophan to acidic environment

or addition of protons to the residues in the immediate

environment of tryptophan. Observed lower Ksv value at pH

1.8 indicated that tryptophan residue becomes more inac-

cessible to solvent in the acid-denatured state. Thus, slight

red shift could be ascribed to protonation of acidic group in

the vicinity of tryptophan residue.

Fig. 3 shows the acid-induced unfolding of asialofetuin

as monitored by ellipticity measurements in the near-UV

region at 256 nm. The CD signals at 256 nm are not

assigned to specific aromatic amino acid residues, but in

this spectral region disulfide bonds have possible contribu-

tions to the CD signals and, nevertheless, they are reflective

of changes in the overall tertiary conformation of the

protein. As can be seen from the figure, there was no

significant change in MRE 256 values from pH 7 to 4;

further decrease in pH leads to a loss in tertiary structure. At

pH 1.8 the loss in tertiary structure was found to be

maximum and MRE value approached to that of GnHCl-

denatured protein (Table 1).

Fig. 4 shows the acid-induced unfolding as monitored by

ANS fluorescence at 470 nm. From the figure, it can be

observed that there was a slight increase in fluorescence

with decrease in pH (upto pH 5.5); further, it remained

constant in the pH range 5.0–2.0. There was maximum

Fig. 3. pH dependence of CD spectra of asialofetuin at 256 nm. Protein

concentration was 30 AM. Experiments were carried out at 25 jC in 10 mM

of the following buffers: pH 1–2.8, glycine-HCl buffer; pH 3–5, sodium-

acetate buffer; pH 6–7, sodium-phosphate buffer.

Fig. 4. pH dependence of ANS fluorescence spectra of asialofetuin at 470

nm. Protein concentration was 3 AM and the experiment was carried out at

25 jC in 10 mM of the following buffers: pH 1–2.8, glycine-HCl buffer;

pH 3–5, sodium-acetate buffer; pH 6–7, sodium-phosphate buffer.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199 195

ANS binding at pH 1.8, which is suggestive of exposure of

hydrophobic patches as reported in our earlier communica-

tions [9–11].

To ascertain whether the protein state observed at pH 1.8

represented the MG-like state, we compared the far-UV and

near-UV CD spectra, cooperativity of unfolding and ANS

binding of asialofetuin at pH 1.8 with those obtained at pH

7. Fig. 5 shows the far-UV CD spectra of asialofetuin in the

native state (curve 1), acid-unfolded state (curve 2) and

completely denatured state in the presence of 6 M GnHCl

(curve 3). The curve for the native state has two minima,

Table 1

Summary of different spectral properties of asialofetuin

Variable Native Acid-

denatured

state

GnHCl-

denatured

state

MRE at 222 nm � 14,800 � 10,300 � 2000

MRE at 256 nm � 45.5 � 10 � 5.7

% Helix 47 29 –

Relative fluorescence

intensity at 340 nm

(excitation at 280 nm)a

100 137 120.9

Relative fluorescence

intensity at 340 nm

(excitation at 296 nm)a

100 111.8 125

kmax (when protein was

excited at 280 nm)

328 340 356

kmax (when protein was

excited at 296 nm)

328 332 352

ANS fluorescence

intensity

38 92 –

Cooperativity

(thermal transition)

cooperative weakly

cooperative

–

a Fluorescence of native protein was taken as 100%.

one at 208 nm and the other at 222 nm, characteristic of a-

helical structures determined from MRE value at 222 nm by

the method of Chen et al. [21]. The spectra of the acid-

unfolded state retained all the features of secondary struc-

ture, although there was a decrease in the MRE values at

222 nm (Table 1), indicating a loss of about 32% a-helical

structure from native state. Asialofetuin in the presence of 6

M GnHCl lost all the features of secondary structure. Fig. 6

shows the near-UV CD spectra in the range 300–250 nm for

Fig. 5. Far-UV CD spectra of asialofetuin in the native state (—) at pH 7, in

acid-unfolded state (- - -) at pH 1.8 and completely denatured state (. . .) in6 M GnHCl. Spectra were recorded in the wavelength region 200–250 nm.

Experimental conditions were similar as stated in legend for Fig. 1.

Fig. 6. Near-UV CD spectra of asialofetuin in the native state (—) at pH 7,

in acid-unfolded state (- - -) at pH 1.8 and completely denatured state (. . .)

in 6M GnHCl. Spectra were recorded in the wavelength region 250–300

nm. Experimental conditions were similar as stated in legend for Fig. 3.

Fig. 7. Fluorescence emission spectra of ANS bound to native asialofetuin

at pH 7 (—) acid-unfolded asialofetuin (- - -) at pH 1.8 and completely

denatured asialofetuin (. . .) in 6 M GnHCl. Excitation wavelength was

380 nm. Protein/ANS were in 1:100 molar ratio and experiments were

performed at 25 jC.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199196

the native state (curve 1), the acid-unfolded state (curve 2),

and in 6 M GnHCl (curve 3). As can be seen from the

figure, acid-unfolded state lost almost all of its tertiary

structure and resembled more to the GnHCl-denatured

spectrum. Fig. 7 shows the fluorescence spectra of ANS

in the 400–600-nm wavelength range in the presence of

native (curve 1), acid-unfolded protein (curve 2) and

GnHCl-denatured protein (curve 3). As can be seen, binding

of ANS to the acid-unfolded state at pH 1.8 produced a large

increase in fluorescence intensity as compared to native

state (Table 1). Thus, retention of some amount of second-

ary structure with complete loss of tertiary structure along

with maximum ANS binding at pH 1.8 is indicative of the

presence of an MG-like state at this pH.

SEC was also performed in order to characterize the MG

state. The MG has an expanded dimension but retains

substantial compactness. Change in molecular size can

easily be detected by SEC. For native protein, a single peak

centered at 77 ml was observed. However, for asialofetuin

treated with acidic pH (pH 1.8) for 3 h before finally loading

on the column, a small but decrease in the elution volume to

74 ml was observed. This indicates that the dimensions of

the molecule have slightly increased under this condition.

The elution volume for completely denatured protein in the

presence of 6 M GnHCl was found to be 66 ml. Thus, the

results indicate a slight increase in the hydrodynamic

dimensions of the protein under acidic pH, which could

be due to the opening up of the tertiary structure as

compared to native state but they were found to be less

than that of completely denatured state.

For further confirmation of the MG-like intermediate

state at pH 1.8, thermal studies were performed. Fig. 8 shows

Fig. 8. Temperature dependence of CD spectrum of asialofetuin. Native

state at pH 7 (–.–) in 20 mM sodium-phosphate buffer; acid-denatured

state at pH 1.8 ( –o– ) in 20 mM glycine-HCl buffer. A protein

concentration of 3 AM was used. Experiments were carried out in the

temperature range 35–95 jC.

Table 2

Ksv values obtained from Stern–Volmer plot

Subject Ksv (M� 1)

Native asialofetuin 8.5

Acid-unfolded asialofetuin 5.2

GnHCl-denatured asialofetuin 14.2

NATA 19.3

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199 197

the temperature-induced unfolding of asialofetuin at pH 7.4

and 1.8 by MRE measurements at 222 nm. The thermal

unfolding of native asialofetuin is a cooperative process. As

can be seen from the figure, the transition started at 55 jC,decreased continuously up to 92 jC and then tailed off. On

the other hand, thermal transition of acid-induced state (pH

1.8) started at a lower temperature, 40 jC, i.e. thermal

transition started earlier in case of acid-denatured protein

and it is less stable than native. Thermal dependence of far-

UV CD signal indicates loosely ordered secondary structure

of acid-induced state as compared to secondary structure of

native asialofetuin. The results show that unfolding of MG

state of asaialofetuin is weakly cooperative. The weakly

cooperative thermal unfolding of the protein at pH 1.8 is

indicative of its MG-like nature. There are similar examples

in many other globular proteins including Equine, Canine,

Cytochrome c and SNase [25–28].

To confirm the microenvironment of the tryptophan

residue, we compared the exposure of the single tryptophan

residue in the native state with that in the acid-denatured

state and guanidine hydrochloride denatured state by fluo-

rescence quenching experiment, using uncharged molecules

of acrylamide as described by Eftink and Ghiron [22]. Fig. 9

shows the Stern–Volmer plot of quenching of fluorescence

by acrylamide in native state, acid-induced state (1.8) and

GnHCl-denatured state of asialofetuin. Results for the

tryptophan analogue NATA are also included as a standard

for complete accessibility to quencher. Table 2 shows the

Stern–Volmer constant fitted to the linear part of the curve.

Ksv for the native state (8.5) was found to be slightly higher

Fig. 9. Stern–Volmer plot of acrylamide quenching. Native asialofetuin at

pH 7 (– w – ); acid-denatured asialofetuin at pH 1.8 (–4–) and NATA

(– � – ) and GnHCl (–o–). Values shown are the ratios of fluorescence in

the absence of acrylamide ( F0) to the fluorescence at that concentration of

quencher ( F).

than that for the acid-induced state (5.2), a blue shift in kmax

from 340 to 337 nm also indicated that tryptophan residue in

acid-induced state was less accessible to quenching than in

native state. The Ksv value for the GnHCl-denatured state

and NATA were significantly higher than those for native

and acid-unfolded state, suggesting that tryptophan residue

even in the native state is internal. Taken together, these

results, i.e. the presence of significant amount of secondary

structure, higher magnitude of ANS binding, weak cooper-

ativity in the thermal transition and almost complete loss of

tertiary structure, suggest that the acid-unfolded state of

asialofetuin at pH 1.8 resembles the MG state as defined for

other proteins [10,11,25–31].

The results obtained from the experiments discussed

above and previous reports [11,32] lead us to conclude that

removal of sialic acid from the protein fetuin brings about a

considerable structural reorganization, suggesting the differ-

ent behavior of asialofetuin during acid denaturation. This

conclusion is supported by observations summarized in

Table 3. Far-UV CD spectrum of native asialofetuin has

higher content of a-helix as evident from the spectrum of

the native protein having minima at 208 and 222 nm typical

of a-helix. The essential absence of the 222-nm minimum in

Table 3

Comparison of the conformational properties of fetuin and asialofetuin

Variables Fetuina Asialofetuin

Native Acid-

unfolded

GnHCl-

denatured

Native Acid-

unfolded

GnHCl-

denatured

% Secondary

structure at

222 nmb

100 113 13 100 70 12.5

% Tertiary

structure at

256 nmb

100 38 38 100 22 12

% Tertiary

structure

at 290 nmb

100 55 5 100 100 46

Relative

fluorescence

intensity at

340 nm

(excitation

at 280 nm)c

100 62 51.5 100 137 120.9

ANS

fluorescence

intensity

3 58 – 38 92 –

a Taken from our earlier communication [13].b MRE of the native protein is taken as 100%.c Fluorescence intensity of native protein is taken as 100%.

F. Naseem et al. / Biochimica et Biophysica Acta 1699 (2004) 191–199198

fetuin suggests that the protein has low a-helix as compared

to that of asialofetuin. Further, it was observed that acid and

thermal denaturation does not lead to the destruction of

secondary structure of fetuin; on the other hand, there is

significant loss of secondary structure during acid and

thermal denaturation.

Near-UV CD spectrum of acid-unfolded fetuin at pH 1.8

[11] in the entire range from 250 to 300 nm resembled

more the spectrum of completely denatured fetuin in 6 M

GnHCl, but the near-UV CD spectrum of asialofetuin at pH

1.8 resembled more that of native protein in the region of

tryptophanyl residue (295 nm) while the rest of the

spectrum was similar to that of 6 M GnHCl-denatured

asialofetuin.

When protein was excited at 280 nm, intrinsic fluores-

cence of acid-unfolded fetuin was quenched along with a

red shift of 4 nm; while intrinsic fluorescence at 340 nm of

acid-unfolded asialofetuin was more than that of native

protein along with a red shift of approximately 12 nm.

Intrinsic fluorescence quenching of acid-denatured pro-

teins (fetuin and asialofetuin) by acrylamide is higher than

that of native, suggesting the internalization of the single

tryptophan residue in both cases.

The implications of the results presented in this paper

may prove useful in understanding the effect of sialic acid

on partially folded intermediate, which accumulates in the

folding pathway of a protein. Further, the results of thermal

transitions and acid denaturation monitored by far-UV CD,

presented in this paper, taken together with previous reports

[11,31], unambiguously demonstrate that sialic acid plays a

significant role in the stabilization of secondary structure of

fetuin during acid and thermal denaturation.

Acknowledgements

Facilities provided by A.M.U are gratefully acknowl-

edged. B.A. thanks the Council of Scientific and Industrial

Research, New Delhi for financial assistance. The authors

are also thankful to DST (FIST) for providing lab facilities.

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