TA

BMa

b

c

d

a

ARRAA

KHDMU

1

tphsh

rtisdsb

(

h0

International Journal of Biological Macromolecules 75 (2015) 447–452

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

hermal induced unfolding of human serum albumin isomers:ssigning residual � helices to domain II

asir Ahmada,1, Ghazala Muteeba,1, Parvez Alama, Ankita Varshneya, Nida Zaidia,ohd Ishtikhara, Gamal Badrb, Mohamed H. Mahmoudc,d, Rizwan Hasan Khana,∗

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, IndiaLaboratory of Immunology & Molecular Physiology, Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, EgyptFood Science and Nutrition Department, National Research Center, Dokki, Cairo, EgyptDeanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia

r t i c l e i n f o

rticle history:eceived 16 December 2014eceived in revised form 2 February 2015ccepted 4 February 2015vailable online 11 February 2015

eywords:uman serum albumin

a b s t r a c t

In this study we have investigated the heat induced denaturation of HSA by utilizing spectroscopicapproaches including fluorescence and circular dichroism. Thermal denaturation of N isomer (domainsI–III remain intact), B isomer (loss of helical structure of interdomain contacts) and I state (domain IIintact) was found to be co-operative processes while for F isomer domains unfold non-cooperatively.These finding pointed out that during N–F transition, HSA suffers more structural alterations which arenot localized only to domain III. Loss of secondary structure in the temperature range 20–60 ◦C withouteffecting tertiary structure of N isomer of HSA is mainly due to loss in helical extensions connecting

◦

enaturationolten globulerea

domain I to II and domain II to III. All the four thermally denatured states (60–96 C) of HSA retainedapproximately 50% residual �-helical structures. Near-UV spectroscopy used as a probe for tertiary struc-ture indicated that heat denatured states lost almost all of the tertiary contacts thereby forming moltenglobule like states. Furthermore, our results provide evidence that residual helical structures are mainlylocated in domain II.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Human serum albumin (HSA) is most abundant and versatileransport protein found in plasma and interstitial fluids [1]. Therotein is a 585 amino acid residue monomer that contains threeomologous �-helical domains (I–III) encompassing the completeequence [2]. Each domain contains ten helices, six antiparallel-elix and four-helix subdomains (A and B).

Structural and functional aspects of human serum albumin inesponse to chemical denaturants like urea and GnHCl revealedhe existence of intermediates in the unfolding pathway suggest-ng that there are structural parts in the molecule with differenttabilities, which unfold in steps [3]. HSA is known to undergo

ifferent pH dependent conformational transitions, the N↔F tran-ition between pH 5.0 and 3.5, the F↔E transition (acid expansion)elow pH 3.5, and the N↔B transition between pH 7.0 and 9.0

∗ Corresponding author. Tel.: +91 0571 272038; fax: +91 0571 2721776.E-mail addresses: rizwanhkhan@hotmail.com, rizwanhkhan1@yahoo.com

R.H. Khan).1 These authors contributed equally.

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.02.003141-8130/© 2015 Elsevier B.V. All rights reserved.

[4,5]. It is increasingly recognized that the structure of non-nativestates of the proteins can provide significant insight into funda-mental issues such as the relationship between the sequence of aprotein and its three dimensional structure, the nature of proteinfolding pathways and the stability of proteins. In contrast to thelarge amount of the structural information about native folded pro-teins, however, only a limited number of denatured proteins havebeen examined in detail [6–9]. It is therefore of considerable valueto study a wide range of non-native states.

The thermal denaturation of albumin has also been studied onseveral occasions [10–13]. A loss of �-helix (28%) and gain of �-structure (from 6 to 16%) occur between 62 and 75 ◦C as judgedby CD, IR and laser Raman studies [14]. It has also been shownthat tertiary structure of native HSA was more stable compared tosecondary structure during thermal denaturation [15]. The effectof heat up to 45 ◦C or to 20% of maximum denaturation is fullyreversible whereas 60% reversibility is found after exposure to80 ◦C.

Though acid and chemical induced denaturation of HSA involvespartially structured multiple intermediates, the study could notgive detailed information about the molecular structure of the pro-tein. An intermediate (where domains are still intact, but with

4 Biolog

cctrTpdhfappdbtw

2

2

gw

2

bw(rmd

2

cm

2

sD1aer

2

litsbstp

48 B. Ahmad et al. / International Journal of

onformational changes) is yet to be trapped and its unfoldingharacteristics are yet to be studied. Hence, attempts were madeo study thermal unfolding of HSA as a function of pH using fluo-escence, far UV–CD and near spectroscopic techniques as probes.he unfolding of protein by heat has received greater attention, asartially folded residual structure has been found to accumulateuring unfolding process of a number of proteins [16–18]. Thereas been a lot of interest in characterizing these residual structures

or gaining an insight into the possible determinants of the mech-nism of protein folding. Human serum albumin is a multi-domainrotein and its domains are capable of unfolding and refolding inde-endently, but how the unfolding and/or separation of a particularomain affect the stability of rest of the domain(s) remains is yet toe investigated. Therefore, in view of this, we aimed to investigatehermal induced unfolding of N, B, F isomers and I state of HSA,hich display characteristic structure and domain status of HSA.

. Materials and methods

.1. Materials

Human Serum Albumin (essentially free of fatty acid), urea andlycine were from Sigma Aldrich, India. All of the other reagentsere of analytical grade.

.2. Preparation of different states of HSA

Stock solution of HSA (250 �M) was made in 10 mM phosphateuffer pH 7.4 which exist in N form. The B and F isoforms of HSAere prepared by mixing 20 �L of N isoform with 980 �L of pH 9.0

10 mM glycine–NaOH) and pH 3.5 (10 mM sodium acetate) buffersespectively. The I conformational states of Human Serum Albu-in were produced by mixing 20 �L of HSA N form with 980 �L of

esired volume of stock urea solution (10 M).

.3. Protein concentration determination

The protein concentration was determined spectrophotometri-ally using E1%

1 cm of 5.3 at 280 nm on a Hitachi Spectrophotometer,odel U-1500 [19,20].

.4. Fluorescence measurements

Fluorescence measurements were performed on a Shimadzupectrofluorometer, model RF-540, equipped with a data recorderR-3. The fluorescence spectra were measured at 25 ◦C with a

cm path length cell. The excitation and emission slits were at 5nd 10 nm, respectively. Intrinsic fluorescence was measured byxciting the protein solution at 295 nm and emission spectra wereecorded in the range of 300–400 nm.

.5. Circular dichroism (CD) measurements

CD measurements were carried out with a Jasco Spectropo-arimeter, model J-720 equipped with a microcomputer. Thenstrument was calibrated with D-10-camphorsulfonic acid. All ofhe CD measurements were made in 20–96 ◦C range with a thermo-tatically controlled cell holder attached to Neslabs RTE-110 water

ath with an accuracy of ±0.1 ◦C. Spectra were collected with scanpeed of 20 nm/min and response time of 1 s. Each spectrum washe average of 4 scans. Far and near UV CD spectra were taken atrotein concentrations of 5 and 20 �M with 0.1 and 1.0 cm path

ical Macromolecules 75 (2015) 447–452

length cells, respectively. The results were expressed as MRE (meanresidue ellipticity) in deg cm2 dmol−1 which is defined as

MRE = CD [mdeg]10nlCp

(1)

where n is the number of amino acid residues (585), l is the pathlength of the cell, and Cp is mole fraction. Helical content was calcu-lated from the MRE values at 222 nm using the following equationas described by Chen et al. [21,22]

% alpha-helix = MRE222 nm − 234030300

× 100 (2)

2.6. Thermal unfolding of different conformational states of HSA

Temperature induced unfolding of N, B, F isomers and I state ofHuman Serum Albumin was followed by far-UV CD in the range250–200 nm and near-UV CD in the range 300–250 nm. Temper-ature was continuously varied from 20 to 96 ◦C at a constant rateby carefully adjusting the heat control of the water bath. Sampleswere allowed to equilibrate for 1 min at the desired temperature.Reversibility was checked by rapidly cooling the heated sample at96 ◦C to the initial lower temperature.

3. Results and discussion

3.1. Characterization of different conformational states of HSA

CD spectroscopy is valuable tool in biophysics and structuralbiology to understand the conformational changes in proteins andnucleic acids [23–25]. Different isoforms of HSA at pH 7.0, 9.0, 3.5and in the presence of 10 M urea (pH 7.0) represented the N, B,F isomers and I state respectively as reported previously. The CDspectrum of HSA at pH 7.0 has two minima, one at 208 and the otherat 222 nm, characteristic of �-helical structure [26,27]. The N, B, Fand I conformational states of HSA contained around 58%, 55%, 49%and 40.4% �-helical structure (supplementary Table 1) as deter-mined by the method of Chen et al., which is in agreement with thevalues reported by other investigators [28,29]. Information aboutthe tertiary structure of protein was obtained from near UV–CD. Thenear UV–CD spectrum for native state (pH 7.0) showed two minimaat 262 and 268 nm and shoulders at 275 nm and 290 nm, character-istic of di-sulfide bond and aromatic chromophores [5]. We observethe increase in the MRE at 262 nm on increasing the pH of HSAsolution from 7.0 to 9.0 (supplementary Table 1). Alteration in con-formation of domain I results in tertiary structure isomerizationduring N↔B. There was an increase in MRE between 280 nm and250 nm and decrease between 300 nm and 280 nm which denotesloss in tertiary structure at pH 3.5 which is also in accord withthe changes in the secondary structure (supplementary Table 1).In view of previous results showing unfolding of domain III dur-ing N↔F transition, loss of secondary and tertiary structure may beattributed to unfolding of domain III. In the presence of 10.0 M urea,the near-UV CD spectra of HSA also show significant alteration intertiary structure. These changes in tertiary structure may be due tothe unfolding of domain III and partial loss of native conformationof domain I as described previously [28].

As shown in Fig. 1 and (supplementary Table 1) on increasingthe pH of HSA solution from 7.0 to 9.0 there was a blue shift of 4 nmand slight decrease of FI at 340 nm which indicates internalizationof tryptophan in the nonpolar environment. The slight quenchingof tryptophan fluorescence might be due to deprotonation of lys-199 and 195, which are located at a distance of 3.7 A and 7.4 A from

Trp-214 in crystal structure of HSA. Wavelength maximum of flu-orescence of HSA shifted from 340 nm at pH 7.0 to 334 nm at pH3.5 which was reported previously for N↔F transition. Which couldattributed to the reason that HSA contains only one tryptophan in

B. Ahmad et al. / International Journal of Biolog

Fm

doIlob

3

o

ig. 1. Intrinsic fluorescence spectra of N, B, F, and I states of human serum albumineasured by exciting the protein samples at 280 nm.

omain II, the absence of any significant change in tryptophan flu-rescence of HSA in 10 M urea indicated that unfolding of domainI did not occur in N↔I transition. This has also been reported ear-ier suggesting non-involvement of domain II in N↔I transition byther probes [30]. The loss of secondary and tertiary structure maye attributed to unfolding of domain III and/or I.

.2. Temperature induced unfolding of different states of HSA

Figs. 2A, B and 3A show far-UV spectra in the range 250–200 nmf N, B and F isomers of HSA at 25 ◦C, 90 ◦C and thermally reversed

Fig. 2. Effect of temperature on the secondary structure of

Fig. 3. Effect of temperature on the secondary structure of

ical Macromolecules 75 (2015) 447–452 449

spectra at 25 ◦C. Far UV CD spectra of I conformational state ofHSA at 25 ◦C, 76 ◦C and thermally reversed spectra at 25 ◦C wererecorded in the region 250–215 nm (Fig. 3B). The spectra were notrecorded below 215 nm, because in the presence of urea or GnHCl,dynode voltage increased significantly and made the data unreli-able. The intensity of all the states of HSA at 222 nm and 208 nmdecreased at 90 ◦C suggesting a loss in �-helix structure of theprotein. Temperature induced unfolding of all the four states ofHSA was found to be almost completely reversible as shown inFigs. 2 and 3, since sample heated at 90 ◦C showed reversal of the CDsignal when brought back to 25 ◦C. Thermal reversibility was foundto be time dependent, as complete reversibility was achieved onlywhen denatured protein was kept at 25 ◦C for at least 1 h (data notshown).

Fig. 4 shows the temperature induced unfolding transitionsof the four states of HSA as monitored by MRE measurementsat 222 nm. Alterations of MRE at 222 nm are a useful probe forvisualizing varying �-helical contents. Unfolding transitions ofN, B, and I states were found to be cooperative while F iso-mers unfolded non-cooperatively as they lacked the pre-transitionregion. The thermal transitions monitored by ellipticity measure-ments at 222 nm showed the presence of the residual secondarystructures even at 96 ◦C, indicating extreme stability of secondarystructure.

The N, B and F isomers retained around 32%, 29% and 29% �-helical contents at 96 ◦C whereas at 25 ◦C their �-helical contentswere found to be 58%, 55% and 49% respectively. On the other hand Iconformation of the HSA retained 20% �-helices at 76 ◦C comparedto 40% at 25 ◦C. The % residual helical content of all the four stateis around 50%, indicating unusual stability of secondary structure

in some domain(s). As discussed above, it was known that I stateretained domain II intact, the unfolding of I state was attributedto unfolding of domain II [28]. Therefore, it appeared that resid-ual helices might occur in domain II. Very high stability of this

HSA (A) N isomer (pH 7.0) and (B) B isomer (pH 9.0).

HSA (A) F isomer (pH 3.5) and (B) I state (5 M urea).

450 B. Ahmad et al. / International Journal of Biolog

Fi

dd

3

U

ig. 4. Heat denaturation profile of different conformational states of HSA as mon-tored by MRE measurements at 222 nm.

omain compared to rest of the molecule had also been shownuring chemical denaturation [7].

.3. Near-UV CD

To study tertiary structural changes in different in proteins nearV–CD is an important tool [31,32]. CD spectra in the near UV region

Fig. 5. Effect of temperature on the tertiary structure of H

Fig. 6. Effect of temperature on the tertiary structure of H

ical Macromolecules 75 (2015) 447–452

(300–250 nm) provided information about the tertiary structure ofthe protein. It was observed that by increasing the temperature ofdifferent states of HSA up to 90 ◦C, there was an increase in theellipticity between 285 and 250 nm with loss of CD signal at 262and 268 nm (Figs. 5 and 6).

As can be seen from the figure when samples heated at 90 ◦Cin case of N, B and F isomers of HSA and 76 ◦C in case of I statewere brought to 25 ◦C, almost complete reversal of CD signal wasobserved, but the spectra had no characteristic features of thesestates at 25 ◦C. Thermal denaturation profile of N and B isomersmonitored by ellipticity measurements at 262 nm showed the pres-ence of some residual tertiary structure even at 96 ◦C while greaterextent of loss of the tertiary contacts was observed in case of F andI states of HSA (Fig. 7).

3.4. Characterization of unfolding transitions

Figs. 8 and 9 show overlap of the fraction denatured (Fd) of N,B, F and I states of HSA. As can be seen from the figure in the tem-perature range between 20 and 60 ◦C, N isomer of HSA displayedaround 20% reduction in helical contents without affecting tertiarystructural contents of the protein as reported earlier [33]. On theother hand in the same temperature range no alterations of sec-ondary or tertiary structure in case of B isomer was observed. Thismight be due lack of lacked interdomain helices (helical extensionsconnecting domain I to domain II and domain II to domain III) in

B isomer. The loss of secondary structure in N isomer between 20and 60 ◦C might be due to unfolding of these helices.

Fds of N, B and I state of HSA analyzed by tertiary and secondarystructure probes are not overlapping which provides evidence that

SA (A) N isomer (pH 7.0) and (B) B isomer (pH 9.0).

SA (A) F isomer (pH 3.5) and (B) I state (5 M urea).

B. Ahmad et al. / International Journal of Biological Macromolecules 75 (2015) 447–452 451

Fig. 7. Heat denaturation profile of different conformational states of HSA as monitored by MRE measurements at 262 nm.

7.0)

tt((se

Fig. 8. Normalized heat induced transitions curves of HSA (A) N isomer (pH

emperature induced unfolding of these conformational states arewo states processes. It deciphered that large fraction of far UV CD0.55 for N isomers and 0.36 for B isomers) in the transitions region

∼82 ◦C) did not originate from N and B isomers at 25 ◦C, but from apecies that had lost its tertiary contacts completely. These providevidence that N and B isomers convert to intermediate states having

Fig. 9. Normalized heat induced transition curves of HSA (A) F isomer (pH 3.5) and (B

and (B) B isomer (pH 9.0) by measurements of MRE at 222 nm and 262 nm.

significant amount of helical structure without any specific tertiarycontacts around 82–96 ◦C temperatures. In case of F isomers no suchnon-coincidental transition was noted. But in view of significant

residual secondary structure and the absence of tertiary contacts indenatured F isomer, unfolding of F isomers cannot be approximatedas a two state process.

) I state (5 M urea) monitored by measurements of MRE at 222 nm and 262 nm.

4 Biolog

4

orooktoasbrads

pl

A

aNawoR

R

[

[[

[[

[[[[[[

[[[

[

[

[[[

[[

52 B. Ahmad et al. / International Journal of

. Conclusion

Temperature induced unfolding of N, B and I states of HSA is co-perative process, while unfolding of F isomer is a non co-operativeeaction. Thermal induced unfolding processes of all the four statesf HSA are almost completely reversible. Interestingly, processesf refolding are time dependent, because heat denatured proteinept at 25 ◦C takes at least an hour to regain its full structure. Inhe temperature range 20–60 ◦C, ∼20% loss of secondary structuref N isomer is observed without any loss of tertiary structure. Thebsence of this behavior in B isomer suggests that helical exten-ion between domains II and I is the first structure to be affectedy temperature. Heat denatured (96 ◦C) N, B, F, I states of HSAetain residual �-helical structure, while tertiary structures are lostlmost completely. Residual helical structure may be located inomain II, because unfolding of I state (domain II intact) also showsimilar amount of �-helical content.

This study may provide a basis for drug design as itossess binding sites for a variety of exogenous and endogenous

igands.

cknowledgements

Facilities provided by A.M.U are gratefully acknowledged. B.And P.A thanks the Council of Scientific and Industrial Research,ew Delhi, Govt. of India for financial assistance. The authors arelso thankful to DST (FIST) for providing lab facilities. The authorsould like to extend their sincere appreciation to the Deanship

f Scientific Research at king Saud University for its funding thisesearch group NO (RG-1435-019).

eferences

[1] I. Petitpas, T. Grune, A.A. Bhattacharya, S. Curry, J. Mol. Biol. 314 (2001) 955–960.

[

[

[

ical Macromolecules 75 (2015) 447–452

[2] X.M. He, D.C. Carter, Nature (1992).[3] M.K. Santra, A. Banerjee, O. Rahaman, D. Panda, Int. J. Biol. Macromol. 37 (2005)

200–204.[4] T. Peters Jr., All About Albumin: Biochemistry, Genetics, and Medical Applica-

tions, Academic Press, 1995.[5] M. Dockal, D.C. Carter, F. Rüker, J. Biol. Chem. 275 (2000) 3042–3050.[6] B. Ahmad, M.Z. Ahmed, S.K. Haq, R.H. Khan, Biochim. Biophys. Acta 1750 (2005)

93–102.[7] S. Muzammil, Y. Kumar, S. Tayyab, Proteins 40 (2000) 29–38.[8] L. Bian, X. Ji, Distribution, Transition, PLoS ONE 9 (2014) e91129.[9] A.L. Pey, Biochimie (2014).10] M. Iosin, V. Canpean, S. Astilean, J. Photochem. Photobiol. A: Chem. 217 (2011)

395–401.11] R. Yadav, B. Sengupta, P. Sen, J. Phys. Chem. B 118 (2014) 5428–5438.12] G. Sancataldo, V. Vetri, V. Foderà, G. Di Cara, V. Militello, M. Leone, PLoS ONE 9

(2014) e84552.13] W. Li, P. Wu, Soft Matter 10 (2014) 6161–6171.14] N. Samanta, D.D. Mahanta, S. Hazra, G.S. Kumar, R.K. Mitra, Biochimie 104

(2014) 81–89.15] P.L. Privalov, Adv. Protein Chem. 35 (1982) 1–104.16] Y. Moriyama, N. Kondo, K. Takeda, Langmuir 28 (2012) 16268–16273.17] S. Tripathi, G.I. Makhatadze, A.E. Garcia, J. Phys. Chem. B 117 (2013) 800–810.18] B.E. Bowler, Curr. Opin. Struct. Biol. 22 (2012) 4–13.19] K. Wallevik, J. Biol. Chem. 248 (1973) 2650–2655.20] A. Varshney, Y. Ansari, N. Zaidi, E. Ahmad, G. Badr, P. Alam, R.H. Khan, Cell

Biochem. Biophys. 70 (2014) 93–101.21] Y.-H. Chen, J.T. Yang, H.M. Martinez, Biochemistry 11 (1972) 4120–4131.22] B. Ahmad, S. Parveen, R.H. Khan, Biomacromolecules 7 (2006) 1350–1356.23] J.M. Khan, A. Qadeer, E. Ahmad, R. Ashraf, B. Bhushan, S.K. Chaturvedi, G. Rab-

bani, R.H. Khan, PLoS ONE 8 (2013) e62428.24] S.K. Chaturvedi, E. Ahmad, J. Khan, P. Alam, M. Ishtikhar, R. Khan, Mol. Biosyst.

11 (2015) 307–316.25] M. Shakir, M.S. Khan, S.I. Al-Resayes, U. Baig, P. Alam, R.H. Khan, M. Alam, RSC

Adv. 4 (2014) 39174–39183.26] D.C. Carter, J.X. Ho, Adv. Protein Chem. (1994).27] N. Zaidi, M.R. Ajmal, G. Rabbani, E. Ahmad, R.H. Khan, PLoS ONE 8 (2013) e71422.28] B. Ahmad, M.K.A. Khan, S.K. Haq, R.H. Khan, Biochem. Biophys. Res. Commun.

314 (2004) 166–173.29] S. Era, M. Sogami, J. Pept. Res. 52 (1998) 431–442.30] N. Tanaka, H. Nishizawa, S. Kunugi, Biochim. Biophys. Acta 1338 (1997) 13–20.

31] S.K. Haq, S. Rasheedi, R.H. Khan, Eur. J. Biochem. 269 (2002) 47–

52.32] P. Attri, P. Venkatesu, A. Kumar, Phys. Chem. Chem. Phys. 13 (2011) 2788–

2796.33] B. Ahmad, R.H. Khan, Arch. Biochem. Biophys. 437 (2005) 159–167.