Mendeleev
Communications
Mendeleev Commun., 2020, 30, 211–213
Albumin aggregation promoted by protoporphyrin in vitro
Natalya Sh. Lebedeva, Elena S. Yurina, Yury A. Gubarev,* Aleksey N. Kiselev and Sergey A. Syrbu
G. A. Krestov Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo,
Russian Federation. E-mail: yury.gu@gmail.com
DOI: 10.1016/j.mencom.2020.03.027
Protoꢀorꢀhyrin
Protoporphyrin upon its binding with serum albumin
changes its secondary structure due to the conversion of part
of a helices into b-folding. This process results in the
association of albumin globules in vitro.
Aggregation
Keywords: porphyrin, albumin, aggregation, UV-VIS spectroscopy, IR spectroscopy, fluorescence.
Protoporphyrin belongs to the blood group porphyrins and its
complex with Fe2+ called hemin represents a prosthetic group for a
number of proteins including those involved in transport and storage
of oxygen (e.g. hemoglobin, myoglobin), electron transfer, drug and
steroid metabolism (e.g. cytochromes) as well as signal transduction
(e.g. nitric oxide synthase, guanylate cyclase). The content of
protein-free hemin in a body is controlled by hemoxygenase.1
Lack of heme or problems in the synthesis of protoporphyrin result
in severe diseases such as anemia and porphyria.2 For their
treatment, drugs containing synthetic hemin3,4 are typically used.
On the other hand, an excess of protein-free porphyrin also leads
to various pathologies, including temporary thrombosis, liver failure
and hemorrhagic diathesis.5,6 Moreover, an excessive amount of
hemin may cause lysis of erythrocyte membranes especially in
sickle-shape cell anemia,7 oxidation of low-density lipoproteins8
and formation of fatty acid hydroperoxides. These processes lead
torenalfailureassociatedwithintravascularhemolysis, hemorrhagic
damage of the central nervous system and atherogenesis.8
Localization as well as concentration of protoporphyrin and
heme should be strictly regulated in an organism, since any
deviation from their optimal amount causes a pathological condition.
In turn, the porphyrin content is influenced by the effectiveness of
enzymes catabolizing heme as well as by the efficiency of transport
systems that ensure the circulation of hydrophobic porphyrin in
cells, intercellular fluid and bloodstream. Some systems for the heme
andprotoporphyrin transportation in mammals have been discovered
and extensively explored.
The data on the transport proteins causing the passage of
tetrapyrrole macroheterocyclic compounds through the cell
membrane, such as HCP1 proteins, FLVCR, Abcg2 and Abcb6,
as wellasofextracellularheme-bindingproteins,suchashemopexin,
haptoglobin and serum albumin, is summarized in the review.9
According to the results of our works10–12 on the interaction of exo-
genous synthetic macroheterocyclic compounds with serum albumin,
it was hypothesized that the interaction of endogenous porphyrins
with albumin can lead to the protein aggregation. This work was
aimed at the experimental verification of the hypothesis about the
protein aggregation mechanism. The acquired results can afford
a detection of the molecular-level reason for b-folding of globular
proteins, which is an ‘identity card’ for diseases like amyloidosis
and hypoalbuminemia.13
The investigation of the interaction of hydrophobic proto-
porphyrin 1 with BSA† was carried out in a medium containing
0.5 m NaCl with DMF added to the concentration not exceeding
0.19 m. The synthesis of protoporphyrin 1 is shown in Scheme 1.
The chosen concentration of NaCl eliminated the effect of poly-
electrolyte protein swelling, while DMF provided the solubility
†
Bovine serum albumin, fraction V (BSA) was purchased from Acros
Organics (USA).
Protoporphyrin IX dimethyl ester 1. Hemin (5.0 g, 7.6 mmol) and
pyridine (5.0 ml) were placed in a three-necked flask, then MeOH (300 ml),
CH2Cl2 (300 ml) and Mohr’s salt (20.0 g, 51 mmol) were added. Acetyl
chloride (150 ml, 2.1 mol) was gradually added under stirring and cooling,
while the temperature was kept below 35°C. The mixture was stirred for
1 h and then diluted with H2O (500 ml). The bottom organic layer was
separated, washed with aqueous ammonia (25%, 50 ml), then with H2O
(200 ml) and dried over anhydrous Na2SO4. The product was purified by
chromatography on silica gel 60 mesh using CH2Cl2 as the eluent. Yield
4.48 g (99%).
UV-VIS and fluorescence spectra were recorded using an AvaSpec-2048
spectrophotometer (Avantes, Netherlands) at 25ºC. A monochromatic LED
UVTOP-295 (Sensor Electronic Technology, USA) was employed as an
excitation light source in the fluorescence experiments. IR spectra were
recorded on an Avatar 360 IR-Fourier spectrometer (Thermo Nicolet, USA)
in KBr pellets.
Cl
HN
N
N
N
N
N
N
Fe
i
NH
OMe
OH
O
O
O
O
OMe
OH
1
Scheme 1 Reagents and conditions: i, MeOH, pyridine, CH2Cl2, Mohr's
salt, AcCl.
© 2020 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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