Indi an Journal of Bi oc hemistry & Biophys ics
Vol. 39. October 2002 . pp. 3 1R-324
Molten globule intermediates of human serum albumin
low concentration of urea
In
B K Muralidhara and V Prakash*
Departmcnt of Protein Chem istry and Tcchno logy, Central Food Technologica l Research InstilUt e, M ysore 570013 , India
R('c('ived 14 O('celllbe r 200 1; revised (/JIIlllcc('p led II May 2002
Interacti on of non-elec trol y tes such as urea w ith protein s espec ially at lowe r co ncentrati ons is open ing- up newc r co ncep ts in th e understanding of prot ein stabi lit y and folding in protcomi cs. In thi s stud y. th e seco ndary and tert iary stru ctural
characteri sti cs and thermal stabilit y of human serum albumin at lower concentration s of urea ha ve bee n monitored. Th e
protein attain s a molten globul e like stru clllre at co nce ntrati on urea be low 2 M. Thi s struct ural state also shows an increa se
in th e a-heli cal co nte nt as co mpared to th e nati ve stat e. A t co nce ntrati ons o f urea above 2 M. human serum album in starts
unfolding. resulting in a three-stat e trans ition w ith two mid point s o f tran siti ons at around 4 M and 7 M urea co ncentration s.
The characteri sti cs o f th e partial ly Fo lded intermediates are di sc ussed w ith respec t to the three co mponent system ana lyses.
Preferential hydrat ion dominates over prei'c rential interacti on at lower co ncentrat ion of urea (up to 2.S M ) and at higher co ncen tration. th e preferenti al interac ti on overtakes preferential hydration in a co mpetiti ve ma nner. Fo rm ati on of structural in termediates at lower co ncen trati on o f urea is hy pothe siz.ed as a general ph enomenon in protei ns and fit s in w ith th e observati on w ith preferential i nteracti on parameters by T im ashelT and co-wor kers in the case o f Iysoz.y me and ri bonuclea <;e at different pH va lues.
Human se rum albumin (HSA) is a 66 kDa sing le
polypeptide protein composed of three homologo us
hel ical domai ns I, and is used as a model protein in
thi s study to understand the interaction of urea at extended lower and hi gher concentrations. The structural state of several protei ns in denaturants has been
dealt in depth for a number of proteins from a both
energetic point of view, as well as th ermodynamic
angle. If one looks at th e abundant treasure of data,
one can see th e energy o f stabilization and denaturation of proteins is a di rect funct ion of concentrati on of
denaturants. Of importanc e are th e observations made
by Timasheff" , whi ch show for th e first time that th ere
are t"vo types of interacti ons poss ibl e with proteins
depending on th e denaturan t concentration ; One at
low co ncentration and! the other at hi gh concentration
of denaturant. The th ermodynamics angl e of such a
phenomena has been looked into both by enth alpi c
measu re ments of HS A at low and hi gh co ncentrati on
of urea throu gh diffelrential scanning ca lorimetry , as
';'Corresponding author
E-ma il: prakash @c ftri .eom
T el: +9 1-R2 1-5 17760; Fax: +9 1- X2 1-S16308
Il bbrel'imi(JIIs: HSA , human serulll albumin: ANS , I-anilinonaphthalene-8-su lfonate: GdnHCI. guanidine hydroc hl oride:
R ase. ribonuclease: T Ill.(app)' apparent thermal tran siti on tempc ralllre.
well as apparent th ermal tran si ti on te mperature measurements substantiated by structural ana lyses. This
throws more light on the mechani sm of interac ti on or
urea at lower co ncentrati on ge neratin g th e molten
globul e like structures and would al so hel p understa nd
the mechani sm of the structural state of protein at
hi gher conce ntratio n of th e denaturant. The stru ctural
stability of HSA at very low and high concentrations
of urea i.e. below 2 M and above 7.5 M, we re studied
exc lusively from th e above point of view.
Most of the preferential interacti on studi es with
proteins were pcrformcd between 2 M to 8 M of urea
and 1 M to 6 M of GdnHCI. The interaction of urea
and GdnHCI with RNase AJ , bovine serum albumin"!
and other proteins5 showed a decrease in net preferential interaction after reac hin g the maximum prefe rential interaction and al so completely unfo lded state.
Anomalous behavior of proteins at 1m\- co ncentration
of denaturants has been reported in a number of cases
where structural intermediates have been characterized6 -8 . However, no emp has is is made on the mechani stic angle of denaturant interaction with proteins.
Our initial study on such an interaction showed that
urea at low concentrati ons preferentially hyd rates
HSA and increases the apparent Stokes radiu s of th e
molecule 9 . Preferential interaction of urea with HSA
was al so shown to dec rease at very hi gh co nce ntra-
MURALlDH ARA & PR AKAS H: MOLTEN GLO BULE INTERM EDI ATES OF HUMAN SERUM ALB UMI N
ti ons (>8 Mt The interacti on of urea at lower co ncentrati ons with proteins in terms of stru cture and stability are less understood as co mpared to th at at
hi gher co ncentrati ons and th e general mec hani sm of
interac ti on of urea at low co nce ntrat ion with protein s
is still not clearl y understood 10.11. The structural in termediates characteri zed at low denaturant concentrati ons diffe r from th e kineti c interm ediates formed
t' Id Ing
'
. o f' Im'
. theo
earIy In
path way I ?-. 13' . Hence, .It IS
port ance to understand th e interacti on of urea especiall y at low co ncentrati ons with HS A direc tl y from
th e calorimetri c meas urements as well as stru ctural
angle of the pro tein in so luti on.
Materials and Methods
Ma terials
Human serum albumin , Co hn frac ti on V (esse ntiall y fatty ac id free), ANS and urea were obtained
fro m Sigma Chemi cal Co. , St. Loui s, Mi sso uri , USA .
The monomeri c form of HSA was prepared by passing through Sephadex G-IOO ge l to remove other
polymers and th e homoge neity of the eluate was establi shed by SOS-PAGE I4. The an alyti cal grade
buffe r salts, triple qu artz di still ed water were used in
all of th e ex periments.
3 19
least 24 hI' with stirrin g at 30 u C. The res pecti ve dialyzates were ll sed as blank s to monitor the therm al denaturati on profil e of equilibrated protein using Gil fo rd
Res ponse-II spectrophotometer. The absorbance at
287 nm was monitored over a temperature range of
25"C to 95°C, with th e temperature increment programm ed fo r 0.6"C/min . Apparent thermal tran si ti on
temperature (Tm.(app) was ca lcul ated according to
standard procedure using also the softw are of Gi Iford
Res ponse-II spectrophotometer l6 .
(ii) Ditlerelliiol scalll1illg colo rillletric lI1 eth od
Oi ffe renti al scanning calorimetri c measureme nts
were made using MC-2 ultrase nsiti ve mi crocal orimeter (Microcal Inc., USA ) at a scan rate of 1.5"C/min .
Instrumental baseline was determined with both th e
cell s filled with degassed dial yzate before sca nnin g of
each sa mpl e. HS A equilibrated either with buffe r or
with urea was degassed and exact conce ntrati on was
determined with appropriate cO ITecti ons for mo lar
ex tin cti on coeffi cients of HS A. A co ncentrati on of 6
mg/ml of HSA was used in all of th e ex periments.
The data was analyzed using Ori gin so ft ware (2.9
Ver. ) and was best fit to non-t wo-sta te model of denaturati on. The thermodynami c param eters were ca lcul ated acco rdin g to th e procedure desc ri bed elsewh ere l 7.
Me th ods
Circllla r dich roic spectrallll eo.wrelllellis
Flu o rescell ce silldies
Far-ultraviolet circul ar dichroic studi es were performed betwee n 200 nm to 260 nm using Jasco J-20C
automati c record ing spec tropo larimeter at 30°C with I
nm bandw idt h.
ear-ultra violet circular dichroic
studi es we re perform ed betwee n 250 nm to 350 nm
using Jasco J-8 10 software dri ve n spectropolari meter
at 30"C withl O nm band width . HSA was equili brated
with varying co ncentrations of urea by equili brium
di alys is for at least 24 hI' with stirring at 30 ± 2"C with
th e co nce nt ra ti on of 2.8x I 0-6 M and 7 .Sx I 0 6 M was
used respec ti ve ly fo r far-UV and near- UV anal yses.
The mean res idue we ight of I 13.57 was used fo r all
calcul ati ons. The data was analyzed accordin g to the
1standa rd procedu re ).
(i) HSA illirillsic flll o rescell ce
Therll/al dCIl{/f llral ioll silldics
(i) Sp eu/"Oscop ic II/ clh od
HSA soluti on co nce ntrati on of 2.8x 10-1> M in di fferent urea co ncentrati on was di alyzed again st large
amount of th e res pecti ve co nce ntrati on of urea for at
A protein of co ncentration of 2.8 x 10-6 M was
equilibrated to required co ncentrati on of ureG by di alys is. The flu orescence emi ss ion properti es of th e
equilibrated protein were monitored in th e ran ge
300-400 nm at 30"C using Shimadz u RF-5000 . pectrofl uori meter 18 .
0"
(ii) /\NS illdllced flu o rescell ce sludies
HSA at co nce ntrati on of 2. 8x 10-6 M was equili brated with different urea co nce ntrati ons an d was
mi xed with fres hl y prepa red A IS. The mixtu re was
incubated with co nstant shaki ng at 100 rpm at 30"C
for 3 hr in Queuc orbital shaker. Fluoresce nce em ission was moni to red between 450-550 n111 kecp ing th e
exc itati on at 395 nm. Onl y ANS with d ifferent urea
co nce ntrati ons and ANS with HSA in bu ller were
used as proper bl anks. AN S:HSA mole rati o of 300: I
was used with varying eq uilibrium co ncentratio n of
urea.
320
INDIA
J. B10C HEM. BIOPHYS. , VOL. 39, OCTOBER 2002
Results and Discussion
Secondary structural analysis is used to monitor the
subtle conformational changes in proteins derived
either from specific and/or non-specific ligand/solvent
in teractions with different structural motifs and/or
domains of proteins. Circular dichroic measurements
of HSA equilibrated to different urea concentration
A
M
22
'0
><
E
c
N
N
N
,.......,
CD
20
~
18
B
0-0-0
-
80
0/
( ,)
0
0
I
c:
0.
~
r
E
/
75
0
/
o
0
1
2
3
Urea [M]
Fig. l- 'A: Changes in th e c irc ul a r di c hro ic spec tral rotations o f
HS A at 222 nm as a fun c ti o n o f lo w conce ntration s o f urea at
300 e [Circu lar dichroi c studi es were perfo rm ed bet ween 200 nm
to 260 nm using lasco 1 20-C a ut o mati c recording spec tropol arimeter at 30"e with I nm band w idth. Ro tati o ns at 222 nm wtre
6
e va lu ated. HSA so luti on conce nt rati o n of 2 .8x 10- M was used for
spec tra l analysis. The mea n residue weig ht of 11 3.57 was used for
2
l
al l calc ul ati o ns. (8) is in deg.cm dmor ): B: Changes in the a ppa re nt th erm al transiti on te mpe rature (T m.(app») of HSA as a fun cti o n o f low concen trat io ns o f urea IThe therm al transiti o ns were
measured to an acc uracy o f ± 0.5°C using th e absorbance at 287
nm o n a G ilford Respo nse-ll th e rmal programming spec trophoIO m e tc rj
were performed and th e rotations at 222 nm are plotted as a functi on of urea concentration, as shown in
Fig. 1A. At lower urea concentration, the a -helical
content of HSA increased and reached a max imum
value of about 10% at 1.2 M urea concentration where
the rotations at 222 nm reaches a maximum value.
Above this concentration, the rotation s gradually decreased with the decrease in a-helix. But the nearultraviolet studies showed only a subtl e decrease in
the tertiary structure up to 2 M of urea concentration
(data not shown).
Effect of secondary structural change induced by
urea at low concentrations on the thermal stabi lity of
HSA was monitored using both spectroscopic and
differential scanning calorimetric meth ods. In Fig. 1B
is shown the effect of di fferent concen,rations of urea
on the Tm.(app) of HSA, A single thermal transition was
observed at low concentration of urea «2 M) which
was monitored for the cumulative effect of tyrosine
and tryptophan at 287 nm . HSA in phosphate buffer
showed a Tm.(app) of 73°C and at low concentration of
urea it increased up to 81 "c at 1.8 M of urea. Above 2
M urea concentrations no thermal trans itio n was observed (Fig. 1B).
Differential scanning calorimetric measurements
were made as an additional probe of t ermal denat uration spectroscopy and also to evaluate the heat capac ity changes of denaturation. In Fig. 2 is show n the
representative differential scanning f ermogram of
HSA in 0.8 M urea (open circ les). The profile was
best-fit to a non-two-state denaturation model , which
is de-convoluted into two clear transitions (dotted
lines), first at 61 °C and the second at n °e. In Fig. 3 is
shown the effect of urea on the t.C p changes of the
two transitions of HSA at different lower concentrations of urea. The t.C p increases and reaches a maximum value at 1.5 M urea concentration after which it
decreases gradually with increase in urea concentration. Above 3 M urea concentration there was only
one transition observed, the mid point of whi ch decreased with the increase in denaturant co ncentration
fo llowi ng typical denaturation process.
At 2 M and 10 M urea concentrations the difference
in the partial molar volume and other spectral parameters of HSA may be same but the extent of interaction are different as shown in our prev ious report 9 .
Thermal den aturati on studies also showed that the
stability of HSA was increased at lower concentrations of urea as reflected in th e increase in Tm.(app) of
HSA . Differential scanning calorimetric data showed
32 1
MURALlDHARA & PRAKAS H: MOLTEN GLOB ULE INTERM EDIAT ES OF HUMAN SERUM ALBUM IN
20
365
-
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:::::
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(J
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a.
360
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t
E
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a
b
0
E
355
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:::::
co
(J
co
350
--
5
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(.)
345
a.
(.)
0
20
.... ..
30
40
50
60
70
80
.
-
..
<I
340
o
90 100
1
the increased stability of HSA as rellected from the
molar enthalpi c valu es at lower co nce ntrati on of
urea, Te mperature incluced tran siti ons o f HSA from
nati ve to denatured state at lower co nce ntrati ons of
urea are broader compared to nati ve protein indicating
less co-operati vity of the fo ldin g interm ediates, which
is a ge neral observation made in case of many
.
~
protein s.
Role of slI ch differenti al e ffect o f urea at low and
hi gh concentrati ons on th e surface properti es of HSA
was interestin g to probe. The hydrophobic clu sters on
the surface of proteins are very sensiti ve to tertiary
stru ctural changes deri ved from specific and nonspecifi c protein interacti ons. Thi s is es peciall y tru e
with a third component such as ANS and has been
ve ry success ful as a hydrophobic fluorophore to
monitor such changes. In Fi g, 4 is shown the interacti on of ANS with HSA as monitored by the increased
fluorescence emi ss ion intensity of ANS at 485 nm
upon binding to protein . ANS al so binds to native
HSA in bufi'er. Chan ges in the ANS tlu oreseenee only
in urea with the increase in co nce ntration are not very
signifi cant. In the case o f HSA equilibrated with urea,
th e ANS flu orescence intensity increased si gnificantl y
with th e increase in denaturant concentrati on reaching
a maximum at 1.5 M o f urea aft er which it started declining.
3
Urea [M]
Temperature CC)
Fig. 2- Rcprescntati ve diffe re nti al scannin g the rm og ram of HSA
at 0.8 M of urca co ncentrati on IThe dat a wcre obtai lied with th e
scan rate of I S 'C/ min and c urvc- fitt c:d to non-twa-state denaturati oll mec hani sm. The Ilrst (0 I"C) an d secolld (72()C) transiti ons
are represcnted by 'a ' and 'b' rcs pec ti vc ly]
2
Fig. 3- Molar e nth alpic changes of the two th ermal transiti ons of
HS A (' a' and 'b') obt aincd fro m the diffcrential scannin g ca lorimetri c ex pcrime nts are show n as a fun cti o n of low co nce ntrati o ns of urea IThe data we rc bes t deconvo luted to give two transiti o ns using non-two state of denatu ra ti o n anal ysis]
200
..........
c ::>
0
(/)
(/)
<{
---E
E c
w
160
l!)
120
ill CO
'<j'
()
C
ill
()
(/)
ill
....
0
::J
LL
C'O
>,
80
:~
(/)
C
ill
c
40
o
1
234
5
6
Urea [M]
Fig. 4-Fluorescence emi ssion intensit y of ANS in prese nce of
HS A at different co ncentrati ons of urea (squares) IThe intensit y at
ze ro denaturant co ncentration represe nts th e ANS binding to HSA
in th e nati ve state. ANS:HSA mol/mol rati o of 300: I was used in
all th e experimellls with frcshl y prepared ANS in bulTe r. ANS
flu orescence in buffer containing different concentration of onl y
urea is show n as co ntrol (c ircles) ]
322
INDIA N J. BIOCHEM. BIOPHYS, VOL. 39, OCTOBER 2002
1.0
0.8
0.6
::J
lL
0.4
0.2
0.0
P
~I
0
2
4
6
8
10
Urea [M]
Fig. 5- Frac tion of unfolded (Fu) HSA as a fun cti on of urea concentration IThe em ission intensi ly of HSA was meas ured at 30"C
with the exc itation maximu m of 285 nm and the values were used
to ca lculate the denatured fracl ionJ
The effec t of varying urea concentrations on the
intrin sic fluorescence properti es of HSA was monitored in orde r to eva luate th e prote in unfo lding. The
flu o rescence e mi ss ion maximum of HSA was kept at
338 nm with the exci tati o n max imum was mo nitored
of 285 nm. Th e fluorescence inte nsity was monitored
at 338 nm as a function of vary ing urea co nce ntrati o n.
The va lu es were used to calc ul ate the fraction of unfolded state (Fu) with a three-state de naturati o n model
and th e res ults are shown in Fig. 5 . The no n-s igmoidal
pattern of fractio n of unfo lded protein as a function of
urea concentratio n indicates the ex istence of unfo lding intermediates at low concentration of urea. Above
2 M co ncentration of urea, th e unfoldin g pattern follows three-state denaturation and reaches a pl atea u at
around 8 M concentration of urea.
Mo lte n g lobul e states of many proteins are characteri zed in differe nt so luti o n co nditi o ns by varying pH ,
temperature, press ure , salts, no n-e lect ro lytes independe ntly and a lso in d iffere nt co mbin ation with these
parameters. This partly unfo lded state opened-up
newe r avenues to understa nd the pro te in o rgani zati onal hierarchi es . A poss ibl e mechani sm was proposed o n the formation of stab le mo lten g lo bule specie of ribonuclease and lysozy me and HSA under
lowe r urea co ncentrat io ns 2 .9 . In case of HS A th e transiti o n from mo lte n g lobul e to denaturati on state is
between 2 M to 2.5 M for urea. Th e mo lte n g lobul e
state is positivel y hyd rated as compared to nat ive state
g iving mo re stability to the prote in ns indi ca ted by
T m.(app) values and al so parti al mo lar volume o f HS A.
ANS, a hydrophobic flu o rescent probe has been used
in many prote in syste ms to characterize th e molten
g lobul e state eithe r in lower concentrati on of denaturant or with differe nt pH. Here , we show th at ANS
binding to HSA reaches a maxim um at 2 M co ncentrati o n of urea wh ere hydrati o n a lso reaches maximum . Since ANS is in aqueo us med ia it can reach the
ex posed hydrophobi c gro ups and bind stro ng ly to
the m . Once the preferential bindin g of urea starts, th e
ANS binding decreases, overridin g all th e c harac te risti cs of th e mo lten g lobul e. HS A has 55 % a-heli x
that can be eas il y perturbed by the c hange in the so lvent e nviro nment (Prakas h and Muralidh ara, unpubli shed res ults) a nd wea k reversible binding of li ga nd s
lik e chl o roge ni c ac id and caffeic aci d to HS A 19. Inc rease of ro tati o ns up to 1500 at 222 om resultin g in
10% inc rease in the a- he lix in th e maximum hydra ti o n reg io n is show n in Fig. 1 A. Fluorescence studi es
also showed th at there is no perturbatio n o f th e s ing le
try ptoph a n (Trp-2 14) located in do main II of HSA at
lower co ncentrati o ns of urea as there was no change
in th e e mi ss ion maximum. However, th e changes in
the tertiary stru cture were subtl e since the HSA
stru cture is mainly dominated by secondary structural
e lements. The mo lte n g lobul e state of proteins are
mainl y c haracteri zed at lower concentratio n of denaturants and there are many repo rts in the lite rature till
recently , showing the molten g lobul e properties like
20
decreased prote in vo lume in case of ribo nuc lease ,
21
increased thermal stability in case of barnase and
poss ib ility of hydrati on of exposed hydrop hobic
groups in case of tubulin22.
However, th e data from Timasheff fo r th e first
time indi cate th at at low concentration of urea, RNase
and ly sozyme have a characteri sti c prefere nti al hydration very simil ar to the data observed in HS A 9 ,
sugges tin g the predo min ance of preferenti a l hydration
of protein s at these lower concentrations of urea. Supporting data are evident from the interac ti o n of model
peptides with urea s ugges tin g favourable inte raction
of mode l peptides at lower concentration of urea2J.
This ce rta inly establishes the di sting ui shing fe ature of
the mechani sm of interac tion of the low and hi gh concentrati o n of urea with HSA . If a mo lec ule ex ists in
two fo rm s, na me ly native and unfo lded, then the effect o f th e added co-solvents wi ll be th e stab ili zntion
of th e native prot e in if the coso lvent is pre fe re nti all y
MURALlDHARA & PRAKASH: MOLTEN GLOBULE INTERM EDI ATES OF HUMAN SERUM ALBU MI N
excluded, of course relative to water from th e protein
surface. But in a solvent like urea, in thi s study , it will
preferentially interact with the unfolded protein and
further increase in urea co ncentrati on decreases the
preferential interac ti on due to steric repulsion and also
affects the structural features of the unfolded protein.
Conclusion
Interaction of urea at high concentrations with several proteins was monitored by partial specific volume
meas urements and calculations of various preferential
interaction parameters2.3.24.25. Many proteins acquire
molten globul e state at specifi c lowe r co ncentrati ons
of urea and recentl y it has been show n in the case of
RNase and lysozy me by Timasheff at different pH
va lues 2. HSA is preferentially hydrated to different
ex tents as th e urea concentration increased up to 2 M
with the hydration max imum at 1.8 M. The preferenti al binding of urea starts with the binding minimum
after sliding through th e stage of maximum preferential hydration as show n in our earlier report'!. The
preferential binding and unfolding region of HSA in
urea, as our results show, is not very sharp when
compared to RNase 25 and wheat germ Iipase 26. The
reason may be due to the more co mpact and stable
structure of HSA with 17 di sulfide bridges. The
binding maximum reaches plateau befo re the much
stand ard 8 M urea concentration, i.e. at 6.5 M. This
has been shown in case of HSA interactions with
aqueous urea and alkylurea by densimetry method 27
and also with the interaction of anions in presence of
urea28.
i ..
The structural stability data from thi s study correlates well with th e preferential hydration phenomena
of HSA <) and RNase2 at low urea concentrations. Thi s
explains one of the possible mechanism for the
anomalous behavior of proteins at low denaturant
conce ntrations whose structure is quite different from
th e kinetic intermediates formed in the early folding
1<) 30 . Th e pnme
.
d'ff
'
stages-·
I erence between the stabl'11zation and destabilization of HSA by urea arises from
the fact th at preferential hydration overrides preferenti al interacti on upto 2.5 M of urea after which the unfolding process would result in preferential interaction
dominating the preferential hyd rati on to bring about
th e negative values of parti al molar volume. As the
protein opens up completely at hi gher concentration
of urea exposing more sites, then th e differential of
th e preferenti al hydrati on and preferenti al interaction
reduces considerably, thu s one sees the fall in prefer-
323
ential interaction values. Thi s emphasizes the signifi cance of molten globule state of proteins at low denaturant concentrations which has uniquely differe nt
properties and apparently more stabl e than the nati ve
protein itself. The formation of stable st ructural intermediates es pecially at low concentrations of denaturants may possibly be a general phenomenon 1Il
several globular proteins.
Wh en we look at preferential binding of urea to
human serum albumin, there is complexity in terms of
both positive and negative ~3 where one cannot directly attribute it just for unfolding of the pro tein
molecul e alone. Such variation in the transfer of free
energy with varying urea concentration is due to th e
unfavourabl e thermodynamic interaction of urea up to
1.5 M and the meas ured free energy of interacti on in
the transition region after 1.5 M which can be a function of the fraction of the production in the native and
denatured proteins in term s of chemical potentials as
being indicated by Timasheff2 for RNase. Lapanje and
co-workers 3l.33 in their examination of preferential
interaction of alkyl urea with ~-lactoglobulin 31 and
myoglobin 32 have shown that alkyl ureas are preferentially excluded at low concentrations. The transfer
enthalpies measured calorimetrically were negati ve
for urea 33 and positive for alkyl urea and has been
interpreted as due to the domination of hydrophobic
nature of the alkyl groups. An excellent interpretation
has been given by Timasheff2 for such a generic observation that the exchangeable sites are characterized
by identical contact enthalpies and have identical exchange affinities and these results certainly would
represent the site occupancy of the protein molec ul e.
This is evident according to Timasheff that the
(Om3)/(Om2) are much smaller than the number of surface sites on the protein molecule as well as number
of sites that the RNase can make intact for their studies . Thi s is true of human serum albumin also and
the (Om:;)/(Om2) we have observed can hav e a similar
interpretation . This rightly indicates that the site occupancy and the thermodynamic binding stoichi ometry carry opposite signs2. However, one should keep
in mind the large number of di sulfide linkages (seventeen) that stabilise the HSA molecule in vivo,
probably in different solution conditions to carryon
its function s.
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