Methemoglobin binding to model phospholipid membranes
The interaction of methemoglobin with model phospholipid membranes composed of phosphatidylcholine and its mixtures with phosphatidylserine or diphosphatidylglycerol has been studied. The binding isotherms have been analyzed in terms of two-dimensional lattice models of surface adsorption and incorp...
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nasplib_isofts_kiev_ua-123456789-1523972025-02-09T21:50:10Z Methemoglobin binding to model phospholipid membranes Зв'язування метгемоглобіну з модельними фосфоліпідними мембранами Связывание метгемоглобина с модельными фосфолипидными мембранами Gorbenko, G.P. Структура и функции биополимеров The interaction of methemoglobin with model phospholipid membranes composed of phosphatidylcholine and its mixtures with phosphatidylserine or diphosphatidylglycerol has been studied. The binding isotherms have been analyzed in terms of two-dimensional lattice models of surface adsorption and incorporation of the protein into the lipid bilayer. The binding parameters including an association constant, binding stoichiometry, enthalpy and entropy contributions to free energy change have been estimated. Исследовали взаимодействие метгемоглобина с модельными фосфолипидными мембранами, состоящими из фосфатидилхолина и его смесей с фосфатидилсерином и дифосфатидилглицерином. Изотермы связывания проанализированы в рамках двухмерных решеточных моделей адсорбции на поверхности и встраивания белка в липидный бислой. Проведена оценка константы ассоциации, стехиометрии связывания, вкладов энтальтиного и энтропийного факторов в изменение свободной энергии при образовании белок-липидных комплексов. Досліджували взаємодію метгемоглобіну з модельними фосфоліпідними мембранами, сформованими з фосфатидилхоліну та його сумішей з фосфатидилсерином і діфосфатидилгліцерином. Ізотерми зв'язування проаналізовано в рамках двовимірних моделей адсорбції на поверхні та проникнення білка в ліпідний бішар. Здійснено оцінку константи асоціації, стехіометрії зв'язування, внесків ентальпійного та ентропійного факторів у змінення вільної енергії при утворенні білок-ліпідних комплексів. 2000 Article Methemoglobin binding to model phospholipid membranes / G.P. Gorbenko // Биополимеры и клетка. — 2000. — Т. 16, № 1. — С. 16-21. — Бібліогр.: 23 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.000551 https://nasplib.isofts.kiev.ua/handle/123456789/152397 577.37 en Биополимеры и клетка application/pdf Інститут молекулярної біології і генетики НАН України |
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Структура и функции биополимеров Структура и функции биополимеров Gorbenko, G.P. Methemoglobin binding to model phospholipid membranes Биополимеры и клетка |
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The interaction of methemoglobin with model phospholipid membranes composed of phosphatidylcholine and its mixtures with phosphatidylserine or diphosphatidylglycerol has been studied. The binding isotherms have been analyzed in terms of two-dimensional lattice models of surface adsorption and incorporation of the protein into the lipid bilayer. The binding parameters including an association constant, binding stoichiometry, enthalpy and entropy contributions to free energy change have been estimated. |
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Article |
| author |
Gorbenko, G.P. |
| author_facet |
Gorbenko, G.P. |
| author_sort |
Gorbenko, G.P. |
| title |
Methemoglobin binding to model phospholipid membranes |
| title_short |
Methemoglobin binding to model phospholipid membranes |
| title_full |
Methemoglobin binding to model phospholipid membranes |
| title_fullStr |
Methemoglobin binding to model phospholipid membranes |
| title_full_unstemmed |
Methemoglobin binding to model phospholipid membranes |
| title_sort |
methemoglobin binding to model phospholipid membranes |
| publisher |
Інститут молекулярної біології і генетики НАН України |
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2000 |
| topic_facet |
Структура и функции биополимеров |
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https://nasplib.isofts.kiev.ua/handle/123456789/152397 |
| citation_txt |
Methemoglobin binding to model phospholipid membranes
/ G.P. Gorbenko // Биополимеры и клетка. — 2000. — Т. 16, № 1. — С. 16-21. — Бібліогр.: 23 назв. — англ. |
| series |
Биополимеры и клетка |
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| first_indexed |
2025-12-01T04:11:55Z |
| last_indexed |
2025-12-01T04:11:55Z |
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1850277696784826368 |
| fulltext |
ISSN 0233-7657. Биополимеры и клетка. 2000. Т. 16. № 2
С Т Р У К Т У Р А И Ф У Н К Ц И И Б И О П О Л И М Е Р О В
Methemoglobin binding to model phospholipid
membranes
G. P- Gorbenko
Kharkov State University
4 Svobody Sq., Kharkiv, 61077, Ukraine
The interaction of methemoglobin with model phospholipid membranes composed of phosphatidylcholine
and its mixtures with phosphatidylserine or diphosphatidylglycerol has been studied. The binding isotherms
have been analyzed in terms of two-dimensional lattice models of surface adsorption and incorporation of
the protein into the lipid bilayer. The binding parameters including an association constant, binding
stoichiometry, enthalpy and entropy contributions to free energy change have been estimated.
Introduction. Model protein-lipid systems are pre
sently widely used to gain insight into the na ture of
interactions between two major membrane consti
t u e n t s — proteins and lipids [1 , 2 ] . Protein compo
nent of these systems is often represented by non-
membraneous water soluble proteins, being capable of
forming complexes with lipids [3 , 4 ] . One of the
proteins employed in such model studies is hemo
globin [5 ] .
Numerous data available in the l i terature a re
indicative of the possibility of hemoglobin binding to
lipid bilayer by means of electrostatic and hydro
phobic interactions [5—7 J. To date , hemoglobin s t ru
cture is well characterized, thus providing a basis for
elucidation of general principles and driving forces of
protein-lipid interactions. One important aspect of the
problem envisaged concerns the factors governing the
thermodynamics of the protein association with a lipid
bilayer.
T h e main goal of the present work was to
examine thermodynamic characteristics of methemo
globin (metHb) complexes with phospholipids. Analy
sis of the binding isotherms in terms of two-dimen
sional lattice models of large ligand adsorption to
membranes allowed to est imate association constant ,
stoichiometry of binding, enthalpy and entropy cont
ributions to the free energy change. As a lipid
component of the model systems studied liposomes
composed of phosphatidylcholine (PC) and its mixtu-
© G. P. GORBENKO, 2000
res vrith phosphatidylserine (PS) and diphospha
tidylglycerol (DPG) were employed.
Materials and Methods . Egg yolk PC, beef heart
D P G and beef brain PS were purchased from Bak-
preparat (Kharkiv, Ukra ine) . Oxyhemoglobin was
isolated from human blood according to [8] and
converted to m e t H b by add ing of potassium ferri-
cyanide with subsequent gel-filtration on molselect
G-25. Liposomes from PC and its mixtures with PS
(3:1, mohmol) and D P G (6:1 , mohmol) were obtained
as follows. Ethanol lipid's solution was evaporated
under vacuo and lipid film was then suspended in 10
mM Tr is -HCl buffer, pH 7.4, to a final lipid concen
tration of 10 m g / m l . l i p id suspension was shaked for
10 min, sonicated for 3 min, 4 °С and centrifuged at
30000 g for 30 min in order to remove multilamellar
vesicles. Liposomes with average diameter of ca. 100
nm, remaining in supernatant , were used in expe
riments. Phospholipid concentration was determined
according to [9 ] . Protein concentration was found
using extinction coefficient E401 = 5 .66-10 5 M _ Icm~
[101.
Results and Discussion. In analyzing the protein
(P) association with lipids (L) one of the following
approaches is commonly used. Within the framework
of traditional approach lipid bilayer is treated as
consisting of discrete protein 's binding sites, each
containing n lipid molecules. In this case binding
curves; a re described by Langmuir isotherm, being
applied to a simple chemical equilibrium: P + nL **
о P - « L . However, as indicated in a number of
16
METHEMOGLOBIN BINDING TO MODEL PHOSPHOLIPID MEMBRANES
studies [11—14] , this approach appears to be inade
quate in examining protein-lipid interactions for the
following reasons. First of all, the protein must be
considered as large ligand, interacting with an ar ray
of binding contacts on a membrane surface and
covering simultaneously n lipid molecules. Additio
nally, adsorption of large ligand on the surface can
lead to the steric constraints depending on the spatial
arrangement of the lipids in the protein-lipid contact
region.
Such peculiarities of the protein interaction with
lipid bilayer have been taken into account in a series
of models, particularly, in the models proposed by
Stankowski [12, 13] . It seems also noteworthy that
there exists an approach, principally differing from
those mentioned above. It based on the consideration
of the protein association with an assembly of lipids,
ra ther than with individual molecules comprising lipid
bilayer, so that protein-lipid interactions a re inter
preted as partit ioning of the protein between aqueous
and membrane phases , characterizing only by par t i
tion coefficient [14] .
In the present paper two-dimensional lattice
models of Stankowski [12, 13] have been used to
analyze quantitatively me tHb binding to liposomes of
various composition. Lipid bilayer was modeled as
hexagonal lattice with structural subunits represented
by lipid molecules. According to the formalism emp
loyed, two limiting cases, corresponding to the l inear
and discoid ligand shape , were considered. Note that
the concept of «ligand shape» means geometrical
ar rangement of binding contacts in the protein-lipid
complex. Adsorption of l inear ligand on the mem
brane surface was described by [13] :
(1)
(2)
where B, F a re the concentrat ions of bound and free
protein, respectively, L is the total lipid concentration,
n is the number of lipid molecules per molecule of
bound protein, Ka is association constant , z is the
lattice coordination number (z = 6) . Assuming that
ligand has a shape of disc the following relationship
was used:
(3)
where a is the parameter of excluded area (a = 3) ,
со = 2v r 3n7# .
Equations (1) — (3) were used to analyze experi
mental da ta obtained for negatively charged lipo
somes, composed of P C mixtures with PS or D P G
Ж 0.10
o.oo 1.20
Fig. 1. The isotherms of methemoglobin binding to liposomes
composed of phosphatidylcholine and phosphatidylserine (3:1,
molrmol). Temperature, °С: / — 4; 2 — 13; 3 — 24; 4 — 31; 5 —
37. Lipid concentration 0.8 mM
0.40-
' 0.30-
5 0.20-
0.10-
0.00
0.00 1.20
Fig. 2. The isotherms of methemoglobin binding to liposomes
composed of phosphatidylcholine and diphosphatidylglycerol (6:1,
mol:mol). Temperature, °С: / — 4; 2 — 13; 3 — 24; 4 — 31; J —
37. Lipid concentration 0.8 mM
17
GORBENKO G. P.
(Fig. 1, 2) . In this case it was assumed that the main
type of protein-lipid interactions is the adsorption of
the protein molecule on membrane surface due to
formation of electrostatic contacts. Meanwhile, taking
into account the findings provided by a number of
studies [6, 7, 15] , preferential mode of me tHb
interaction with neutral PC vesicles was supposed to
be the protein penetrat ion into bilayer interior. T h e
refore the binding curves observed for PC liposomes
(Fig. 3) were treated in te rms of the model of protein
incorporation in the membrane [13] :
(4)
(5)
(6)
Parameter n in eqn. (4) corresponds to the
number of lipid molecules occupying surface area
being equivalent to cross-section of the protein part
penetrating in the bilayer.
T h e binding of me tHb to liposomes was exa
mined by monitoring the decrease of protein absor-
0.30
0.00 0.40 0.80
Protein concentration, цМ 1.20
Fig. 3. The isotherms of raethemoglobin binding to liposomes
composed of phosphatidylcholine Temperature, °С: 1 — 4; 2 — 13;
3 — 24; 4 — 31; 5 — 37. Lipid concentration 0.5 mM
bance in Soret band (at wavelength 407 nm) . This
absorbance change is supposed to be a consequence of
metHb structural al terations caused by its interaction
with lipid's hydroperoxides [16] or negatively char
ged phospholipids [17, 18] . According to approach,
developed in the previous studies [20, 21 ], it was
assumed that absorbance decrease (АЛ 4 0 7 ) is pro
portional to the concentration of the bound protein
(B):
(7)
where a is coefficient of proportionality. T h e mea
sured value of ( A i 4 4 0 7 ) (AAr) was corrected for the
light scattering of the protein-lipid mixture using the
following relationship [20] :
(8)
where A0 is the wavelength from the region of
neglijjibly small protein absorbance (A0 = 700 nm) ,
A = 407 nm. Parameter m, determined by the sample
turbidity, was estimated from the plots lg Л vs. lg A
obtained at wavelengths 600—700 nm, according to
equation:
m = (9)
(10)
where PQ
C and P0
e a re calculated and determined
experimentally concentrations of the protein, m is the
number of experimental points.
Presented in Table 1 a re the parameters n and
Ka characterizing me tHb association with liposomes of
various composition. Since the ligand shape is un
known and proves to be irregular, the lower and
upper limits of n were assessed assuming linear or
discoid ligand shape, respectively. Because the cross-
section of me tHb molecule being ca. 2600 A, corres
ponds; to the area of ca. 37 lipid molecules, it seems
likely that real shape of contact region in the protein-
18
METHEMOGLOBIN BINDING TO MODEL PHOSPHOLIPID MEMBRANES
Table 1
Parameters of methemoglobin binding to liposomes derived from the fitting of experimental data to eqns. (l)—(6)
The error of parameter estimation does not exceed 17 % for Ka 20 % for n and 12 % for a
lipid complexes is closer to linear. It should be
emphasized that parameter n accounts for a total
amount of lipid molecules that a re excluded from the
further ligand binding upon the adsorption of one
protein molecule. T h e actual number of protein-lipid
contacts in some cases can be less than n. Such a
situation may take place, for instance, when the
protein binds preferentially to the charged lipid
headgroups, that form clusters upon the protein
association with lipid bilayer [13] . Taking into ac
count this possibility, relatively high n values, derived
for liposomes PC:PS (Table 1) can be interpreted in
terms of preferential me tHb binding to negatively
charged PS molecules. In this case the number of
lipids in contact with the protein (n*) can be eva
luated as nf, where / is the fraction of charged lipid
(f = 0.25). T h e values of n*, obtained in such a way,
would be closer to aforementioned est imates, based
on the protein cross-section and the area of l ipid's
headgroup.
T h e value of association constant (Table 1)
observed at different temperatures were further used
to estimate free energy change (AG) a n d its enthalpy
(AH) and entropy (AS) contr ibutions:
lnK„ = R RT
(11)
(12)
T h e fitting of In Ka plots vs. 1 /T to eqn. (12)
allowed to evaluate AH and AS. As can be seen from
Table 2, the formation of m e t H b complexes with
liposomes is characterized by positive enthalpy and
entropy changes. T h e values of AG a re consistent with
those reported elsewhere for model peptides [19] and
proteins [22] . According to the modern theories of
protein-lipid interactions there exist at least five
factors controlling the thermodynamics of the protein
binding to lipid bilayer, namely i) formation of
electrostatic contacts, ii) hydrophobic effect and al te
rations in iii) the network of hydrogen bonds , iv)
protein entropy and v) lipid order ing [23 ]. As follows
from the theoretical predictions, changes in hydrogen
19
GORBENKO G. P.
Table 2
Thermodynamic parameters of methemoglobin interaction with liposomes
bonding and lipid order ing do not contribute no
ticeably to the total AG value. However, another
factors afore-mentioned may be of significance. T h e
results presented here suggest that the main driving
force of me tHb interaction with lipids is energetically
favourable entropy increase. Th i s , in turn , can be
caused by i) formation of ionic contacts in water, ii)
transfer of nonpolar amino acid sidechains in the
membrane interior and iii) unfolding of the protein
molecule [23] . In this context it seems important to
note that negatively charged lipids, including PS and
DPG, can exert destabilizing influence on me tHb
structure [17, 18] .
Taken together, the results of the present s tudy
indicate that two-dimensional lattice models allow to
obtain reasonable est imates of the binding para
meters , characterizing ei ther surface adsorption or
protein incorporation in the lipid bilayer. From ther-
modynamical viewpoint, entropy factor was found to
play determining role in me tHb association with the
model phospholipid membranes .
Acknowledgements. I thank the referee for the
valuable remarks .
Г. П. Горбенко
Зв'язування метгемоглобіну з модельними фосфоліпідними
мембранами
Резюме
Досліджували взаємодію метгемоглобіну з модельними фос
фоліпідними мембранами, сформованими з фосфатидилхоліну
та його сумішей з фосфатидилсерином і діфосфатидилглі-
церином. Ізотерми зв'язування проаналізовано в рамках дво
вимірних моделей адсорбції на поверхні та проникнення білка
в ліпідний бішар. Здійснено оцінку константи асоціації, стехіо
метрії зв'язування, внесків ентальпійного та ентропійного
факторів у змінення вільної енергії при утворенні білок-
ліпідних комплексів.
Г. П. Горбенко
Связьгсание метгемоглобина с модельными фосфолипидными
мембранами
Резюме.
Исследовали взаимодействие метгемоглобина с модельными
фосфолипидными мембранами, состоящими из фосфатидилхо-
лина и его смесей с фосфатидилсерином и дифосфатидилглице-
рином. Изотермы связывания проанализированы в рамках
двухмерных решеточных моделей адсорбции на поверхности и
встраивания белка в липидный бислой. Проведена оценка кон
станты ассоциации, стехиометрии связывания, вкладов эн-
тальтиного и энтропийного факторов в изменение свободной
энергии при образовании белок-липидных комплексов.
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УДК 577.37
Received 21.07.98
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