Use of Cysteine As a Spectroscopic Probe for Determination of Heme-Scavenging Capacity of Serum Proteins and Whole Human Serum

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Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum Remi Noe, Nina Bozinovic, Maxime Lecerf, Sébastien Lacroix-Desmazes, Jordan Dimitrov

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Remi Noe, Nina Bozinovic, Maxime Lecerf, Sébastien Lacroix-Desmazes, Jordan Dimitrov. Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum. Journal of Pharmaceutical and Biomedical Analysis, Elsevier, 2019, 172, pp.311-319. 10.1016/j.jpba.2019.05.013. hal-02127294

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Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j ournal homepage: www.elsevier.com/locate/jpba

Use of cysteine as a spectroscopic probe for determination of

heme-scavenging capacity of serum proteins and whole human serum

1 1

Rémi Noé , Nina Bozinovic , Maxime Lecerf, Sébastien Lacroix-Desmazes,

∗

Jordan D. Dimitrov

Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006 Paris, France

a r t i c l e i n f o a b s t r a c t

Article history: Heme serves as a prosthetic group of numerous proteins involved in the oxidative metabolism. As result of

Received 19 December 2018

various pathological conditions associated with hemolysis or tissue damage, large quantities of hemopro-

Received in revised form 3 May 2019

teins and heme can be released extracellularly. Extracellular heme has pronounced pathogenic effects

Accepted 5 May 2019

in hemolytic diseases, mediated by its pro-oxidative and pro-inﬂammatory activities. The pathogenic

Available online 7 May 2019

potential of heme is mostly expressed when the molecule is in protein unbound form. The pathological

relevance of free heme deems it necessary to develop reliable approaches for its assessment. Here we

Keywords:

developed a technique based on UV–vis absorbance spectroscopy, where cysteine was used as a spec-

Heme

Hemolysis troscopy probe to distinguish between heme-bound to plasma proteins or hemoglobin from free heme.

This technique allowed estimation of the heme-binding capacity of human serum, of particular heme

Heme-binding proteins

Human serum scavenging proteins (albumin, hemopexin) or of immunoglobulins. The main advantage of the proposed

Absorbance spectroscopy approach is that it can distinguish free heme from heme associated with proteins with a wide range of

afﬁnities. The described strategy can be used for evaluation of heme-binding capacity of human plasma

or serum following intravascular hemolysis or for estimation of stoichiometry of interaction of heme

with a given protein.

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction hemoglobin. As a result of its oxidative potential and prominent

hydrophobicity, free heme is inherently toxic molecule. Likewise,

Heme (Fe-protoporphyrin IX) is a macrocyclic compound heme is considered as endogenous danger signal (alarmin) that

that serves as a prosthetic group of many proteins involved activates different types of immune cells and endothelia, increases

in the aerobic metabolism. By transiently interacting with a vascular permeability, triggers complement cascade activation and

number of intracellular or membrane-associated proteins, heme dysregulates coagulation [3,4]. Accordingly, extracellular hemo-

also participates in the cell signalling and regulation of cellu- proteins and heme contribute to the pathogenesis of diseases such

lar functions [1]. Under physiological conditions most of heme is as malaria, sickle cell disease, sepsis, rhabdomyolysis and other.

intracellularly sequestered. However, release of large quantities In the clinical practice the assessment of hemolysis is performed

of heme-containing proteins (hemoproteins), such as hemoglobin by measurement of the plasma scavenger of hemoglobin, i.e. hap-

and myoglobin can occur as consequence of diverse patholo- toglobin [5]. Thus, a decrease in the plasma concentration of this

gies. Thus, damage of erythrocytes due to genetic abnormalities protein signiﬁes recent hemolytic events. Extensive hemolysis may

of hemoglobin, infections, trauma, or autoimmunity can result also result in overwhelming of scavenging capacity of hemopexin,

in intravascular hemolysis and liberation of massive quantities the plasma protein that binds heme with high afﬁnity [6]. In this

of extracellular hemoglobin [2]. In extracellular compartment case, the extracellular heme can associate with lower afﬁnity to

and upon oxidation, heme relatively easily dissociates from other plasma constituents, including albumin, and lipoproteins

[6,7]. However, as albumin has a slow exchange rate (half-life of

ca. 20 days), recurrent and extensive hemolysis may also result in

∗ saturation of the heme-binding capacity of this abundant plasma

Corresponding author at: INSERM UMRS 1138, Centre de Recherche des Corde-

protein. Since the pathologically-relevant form of heme is the one

liers, 75006 Paris, France.

that is loosely bound to proteins (referred to as free heme), its esti-

E-mail addresses: [email protected], [email protected]

(J.D. Dimitrov). mation is of utmost clinical importance [3]. However, to the best

These authors contributed equally to the work.

https://doi.org/10.1016/j.jpba.2019.05.013

312 R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319

of knowledge, there is no estimation of the total heme-scavenging was used directly. After recording the spectra of the protein solu-

capacity of human serum. There are commercialized techniques for tions, potassium cyanide, glutathione or cysteine were added to

measurement of concentration of total heme in plasma that have the protein solutions at ﬁnal concentration of 10 mM. Following,

been applied in different studies [8–10]. These approaches relay on vigorous homogenization, the spectra were measured as described

colorimetric detection of heme or on the pseudo-peroxidase activ- above.

ity of heme in the presence of hydrogen peroxide. Nonetheless,

these techniques fail to differentiate the hemoprotein-bound from 2.2.3. Determination of cysteine concentration for heme-cysteine

free heme. spectral measurements

Here we describe a simple method that relays on characteris- Cysteine was diluted in PBS to concentrations of 0.156, 0.312,

tic spectral changes of protein-free heme upon interaction with a 0.625, 1.25, 2.5, 5, 10 and 20 mM. The UV–vis spectra of each

thiol-containing compound (cysteine). We demonstrated that this concentration of cysteine in the presence of 20 ␮M hemin were

technique could be reliably used for estimation of the free extra- measured as described above. To obtain differential spectra, the

cellular heme. The method can also be applied for evaluation of the absorbance spectrum in the range 300–700 nm of hemin at 20 ␮M

heme-binding capacity of different proteins and human serum as was subtracted from the spectra of hemin in the presence of each

well as for assessment of quality of heme-binding proteins used in concentration of cysteine.

therapy or as blood substituents.

2.2.4. Building of a calibration curve for heme-cysteine

Hemin was diluted in PBS to concentrations of 0.312, 0.625, 1.25,

2. Material and methods

2.5, 5, 10, 20, and 40 ␮M. The UV–vis spectra of each concentration

in the presence of excess of cysteine (10 mM ﬁnal concentration)

2.1. Materials

were measured as described above. To obtain differential spec-

tra, the absorbance in the range 300–700 nm of heme at a given

Hemin was obtained from Frontier Scientiﬁc, Inc. (Logan, UT).

concentration was subtracted from the spectrum of hemin in the

Cysteine, reduced glutathione, potassium cyanide, DMSO, phenyl-

presence of cysteine. The obtained differential spectra have promi-

hydrazine hydrochloride, 5,5 -dithiobis(2-nitrobenzoic acid), 2,2 -

nent absorbance maximum at 364 nm. The values of the differential

azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), human

absorbance at this maximum were plotted versus the concentration

hemopexin and human hemoglobin were obtained from Sigma-

of hemin.

Aldrich (St. Louis, MO). All chemicals were with the highest

available purity. Human serum albumin (LFB, Les Ulis, France) and

2.2.5. Evaluation of heme-binding capacity of human plasma

human pooled immunoglobulin G (IVIg, Endobulin, Baxter USA)

proteins

were thoroughly dialyzed against PBS and stored before use at

− ◦ Human serum albumin, and human pooled IgG were diluted in

20 C at concentrations of 200 mg/ml and 80 mg/ml, respectively.

PBS at concentrations ranging from 1.25–320 ␮M (dilution by fac-

Human AB serum obtained from healthy donor was purchased from

tor of 2). Alternatively, human hemopexin and human monoclonal

Etablissement franc¸ ais du sang, Paris, France, (ethical authorization

◦ IgG1 (identiﬁed in our previous studies to interact with heme) were

N 12/EFS/079). All stock solutions were freshly prepared and used

diluted in PBS in concentrations ranging 0.312–40 ␮M. Next, 20

within 24 h. Hemin was dissolved in DMSO to ﬁnal concentration

␮M ﬁnal concentration of hemin was added to each protein dilu-

of 2 mM. The stock solutions of the other chemicals and proteins

tion and UV–vis spectra were recorded as described above. After

were always prepared in PBS.

to the same samples was added cysteine at 10 mM ﬁnal concentra-

tion and spectra were recorded immediately. Differential spectra

2.2. Methods

were obtained after subtraction of spectrum of protein-heme from

the spectrum of protein-heme / cysteine at given protein concen-

2.2.1. Settings for spectroscopic measurements

tration. The plotting of the maximum differential absorbance at

If not stated otherwise all measurements of heme interac-

364 nm versus concentration of protein was used for calculation of

tions were performed by using following experimental setting:

the heme-binding capacity of albumin or IgG.

the UV–vis absorbance spectra were recorded by Cary-300 spec-

trophotometer (Agilent Technologies, Santa-Clara, CA) using 1 ml

2.2.6. Evaluation of heme-binding capacity of human serum

quartz optical cells (Hellma, Jena, Germany) with 1 cm optical path.

Serum obtained from healthy blood donor was assayed at dilu-

The spectra were recorded in the wavelength range 300–700 nm

tions (in PBS) in range from 20 to 5120 × (dilution factor of 2). To

with spectral resolution of 1 nm and bandwidth set at 2 nm. The

each serum dilution was added 20 ␮M hemin and spectra recorded

absorbance background derived by the buffer only was subtracted

◦ following intensive homogenization. After the measurement of the

from each reading. All measurements were performed at 25 C.

spectrum of heme in presence of a given dilution of serum, to the

same sample was added 10 mM cysteine, intensively homogenized,

2.2.2. Initial analyses of interaction of heme with low molecular and the UV–vis spectrum recorded. The assessment of serum heme-

weight ligands binding capacity was performed by plotting the value of differential

To identify an appropriate probe that interacts only with absorbance at 364 nm versus the dilution of serum.

protein-free heme, we tested binding of cyanide, glutathione and

cysteine to free heme and protein-bound heme. Hemin was ﬁrst 2.2.7. Detection of free heme under hemolytic condition

diluted to 20 ␮M in PBS. The UV–vis spectra in absence and pres- We used leftover serum samples kindly provided by Dr. Lubka

ence of 10 mM ﬁnal concentration of potassium cyanide, reduced Roumenina (Centre de Recherche des Cordeliers, INSERM U1138,

glutathione or cysteine were recorded. The heme’s ligands were Paris). As a part of this study wild-type C57BL/6 mice were

added immediately before measurement and samples vigorously obtained from Charles River Laboratories. Eight-week-old female

homogenized. C57BL/6 WT mice were injected intraperitoneally with 200 ␮l PBS

␮

For examination of the effects of heme ligation protein bound or same volume of phenylhydrazine (900 mol/kg, corresponding

state 20 ␮M solutions of human hemopexin and human serum to 0.125 mg/g body weight). The blood was collected from the sub-

albumin in PBS were ﬁrst treated with 20 ␮M ﬁnal concentration mandibular vein 24 h after the induction of hemolysis. Serum was

of heme. Human hemoglobin at 5 ␮M (containing 20 ␮M of heme) obtained by centrifugation of blood at 2000 g for 10 min. These

R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 313

Fig. 1. Interaction of low-molecular weight heme’s ligands with protein-bound and free heme. (A) Absorbance spectra of 20 ␮M hemin in the absence or presence of 10 mM

ﬁnal concentration of KCN (blue line), of reduced glutathione (green line) or of cysteine (red line). (B) Absorbance spectra of 5 ␮M human hemoglobin (equivalent of 20 ␮M

heme) in the absence or presence of 10 mM excess of KCN (blue line), reduced glutathione (green line) or cysteine (red line). (C) Absorbance spectra of 20 ␮M human

hemopexin in absence of hemin (gray line) or in presence of 20 ␮M hemin without (dashed line) or with 10 mM excess of KCN (blue line), reduced glutathione (green line)

or cysteine (red line). (D) Absorbance spectra of 20 ␮M human serum albumin in absence of hemin (gray line) or in presence of 20 ␮M hemin without (dashed line) or with

10 mM excess of KCN (blue line), reduced glutathione (green line) or cysteine (red line). All dilutions were performed in PBS pH 7.4. In all cases UV–vis absorbance spectra

of hemin were recorded in the wavelength range 300–700 nm in quartz optical cell with 1 cm path length. The measurements were performed immediately after addition

of the ligands (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article).

experiments were conducted in accordance with the recommen- serum samples containing only apo-HRP and only hemin from cor-

dations for the care and use of laboratory animals provided by responding serum dilutions containing both hemin and apo-HRP.

Charles Darwin ethical committee (Paris, France) and with the eth-

ical authorization from the French Ministry of Agriculture, Paris, 3. Results and discussion

France (APAFIS 3764-201601121739330v3).

Normal mice serum and serum obtained from mice with acute In the present study we aimed to develop a technique for

intravascular hemolysis were ﬁrst diluted 20 in PBS. Then, the detection and quantiﬁcation of protein unbound extracellular

UV–vis spectra of serum samples were recorded in the absence and heme. Such method should be able to distinguish heme-associated

the presence of 10 mM of cysteine as described above. to proteins from free heme in complex systems such as blood

plasma or serum. Interaction of low molecular weight ligands

with heme’s iron leads to considerable changes in the absorbance

spectra of the porphyrin molecule. Hence, low molecular weight

2.2.8. Detection of free heme by apo-peroxidase assay heme-binding substances can be used as spectroscopy probes for

Horseradish peroxidase (HRP) was obtained from Sigma- assessing unbound heme. To discriminate protein-bound from

Aldrich. To prepare apo-HRP, we used the procedure of extraction unbound heme, the probe should change the absorbance spectrum

by acidiﬁed acetone as described in [11]. After dialyses against PBS of unligated heme only. Moreover, the probe ideally should induce

◦

apo-HRP was stored at −20 C until use. distinct spectral changes as compared to those induced by binding

Normal human serum was serially diluted in PBS containing of heme to the abundantly present hemoproteins (hemoglobin and

0.1% Tween 20 in range 10–2560 × (dilution factor of 2). Next, to myoglobin) and plasma heme-scavenging proteins (hemopexin

two identical series of serum dilutions samples we added hemin and albumin).

resulting in a ﬁnal concentration of 10 ␮M. Following, an incuba-

tion of 10 min at room temperature apo-HRP was added resulting 3.1. Selection of spectroscopy probes for assessment of free heme

in 10 ␮g/ml. The enzyme was added to a serum dilutions contain-

ing hemin as well as to a set of identical serum dilutions in absence The interaction of heme with globins, hemopexin and albu-

of heme. The samples were incubated for 10 min at room temper- min involves coordination of heme’s iron by imidazole group of

ature. After the incubation 50 ␮l of each sample was transferred histidine residues [12]. This type of coordination results in charac-

to NUNC 96 well polystyrene plates (Thremo Fisher Scientiﬁc). The teristic bathochromic (red) shifts in the Soret region of the spectra

samples were then mixed with 150 ␮l of reaction buffer - solu- of the oxidized heme. On the contrary, the coordination of heme

tion of 0.5 mg/ml ABTS, in 0.1 M Citrate-phosphate buffer pH 5.0, by thiol-containing compounds such as cysteine residues leads to

containing 6 mM H2O2. The oxidation of ABTS was followed by mea- hypsochromic (blue) shift in the Soret region of UV–vis absorbance

suring the absorbance at 414 nm with a microplate reader (Inﬁnite spectrum [13]. Based on this evidence, we reasoned that thiol-

200 Pro, Tecan). The reconstitution of apo-HRP enzyme activity containing substances could serve as spectroscopy probes that

was evaluated after subtraction of the absorbance at 414 nm of allow discrimination of free heme from protein-bound heme. To

314 R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319

Fig. 2. Quantiﬁcation of the spectral changes of heme following exposure to cysteine. (A) UV–vis absorbance spectra of hemin at concentrations of 20 ␮M were recorded

in absence and presence of 0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 mM cysteine. The differential spectra were obtained after subtraction of the spectrum of hemin in

the absence of cysteine from the hemin spectrum at given cysteine concentration. On the left panel are presented differential spectra. The right panel depicts the cysteine-

concentration dependent increase in the absorbance at 364 nm, a characteristic absorbance for complex of hemin with cysteine. (B) UV–vis absorbance spectra of hemin at

concentrations of 0.625, 1.25, 2.5, 5, 10, 20 and 40 ␮M were recorded in absence and presence of 10 mM cysteine. The differential spectra were obtained after subtraction

of spectrum at given hemin concentration from the spectrum at same hemin concentration in the presence of 10 mM cysteine. On the left panel are presented differential

spectra. The right panel depicts the hemin-concentration dependent increase in the absorbance at 364 nm, a characteristic absorbance for complex of hemin with cysteine.

All dilutions were performed in PBS pH 7.4. In all cases UV–vis absorbance spectra of hemin were recorded in the wavelength range 300–700 nm in quartz optical cell with

1 cm path length. The measurements were performed immediately after addition of the ligands.

test this hypothesis, we ﬁrst measured the UV–vis absorbance spec- time of titration experiment (40–60 min) the cysteine and glu-

tra of oxidized form of heme i.e. hemin in the presence of low tathione stock solutions did no undergo detectable oxidation and

molecular weight heme ligands (Fig. 1A). The free hemin in phos- can serve as reliable spectroscopic probes (Supple. Table 1 and Fig.

phate buffer demonstrated a characteristic absorbance spectrum S2).

with Soret maximum at 388 nm. The addition of excess of high Further, we examined the effects of low molecular heme ligands

afﬁnity ligand of hemin – cyanide resulted in a prominent alter- on spectral characteristics of heme bound to various proteins. The

ation of the absorbance spectrum with the typical red shift of exposure of human hemoglobin to cyanide resulted in a typical red

the absorbance maximum to 423 nm in Soret region and an aug- shift in the Soret region (Fig. 1B). The treatment of the hemoprotein

mentation of the absorbance intensity (Fig. 1A). On the contrary, with reduced glutathione had also a profound effect on the UV–vis

the incubation of hemin with excess of thiol-containing amino absorbance spectrum of hemoglobin. Thus, there was a consider-

acid, cysteine, resulted in a distinctive blue shift in the absorbance able reduction of the prototypic absorbance peak at 405 nm and

maximum to 364 nm, accompanied by an increase in the overall appearance of a novel peak at 364 nm (Fig. 1B). Notably, the incu-

absorbance intensity (Fig. 1A). Hemin-cysteine spectral changes bation of hemoglobin with an excess of cysteine did not induce any

were independent of the solvent used to solubilize hemin before changes in the UV–vis absorbance spectrum (Fig. 1B). Further, we

ﬁnal 100 × dilution in PBS, albeit a lower overall intensity in case studied the interaction of heme with the high afﬁnity heme scav-

of hemin dissolved in NaOH compared with DMSO was detected enger hemopexin (Fig. 1C). As demonstrated previously, interaction

(Fig. S1). The exposure to another thiol-containing substance – glu- of heme with hemopexin resulted in considerable spectral changes

tathione caused less pronounced spectral changes in the UV–vis characterized by a blue shift and a pronounced increase in the

spectrum of free hemin (Fig. 1A). To determine whether thiol- absorbance intensity around 410 nm [14]. The addition of excess of

containing substances (cysteine and glutathione) are chemically cyanide or cysteine, however, did not induce considerable modiﬁ-

stable in PBS solution at pH 7.4, Elman’s assay was applied (see cation of the spectral characteristics of heme-bound to hemopexin.

Supplementary information). We concluded that within the typical Nevertheless, reduced glutathione had a profound effect on the

R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 315

Fig. 3. Evaluation of heme-binding capacity of human plasma proteins. Human serum albumin, human hemopexin, human pooled IgG and human monoclonal IgG1 at

concentrations ranging from 0 to 40 ␮M (320 ␮M in the case of pIgG) were incubated in the presence of 20 ␮M hemin. UV-absorbance spectra were recorded in the absence

and in the presence of 10 mM cysteine. (A) Differential spectra obtained after subtraction of heme-protein spectrum from the corresponding spectrum in the presence of

excess of cysteine. The black lines represent the differential spectra of hemin in the absence of protein. (B) Protein concentration-dependent changes in absorbance maxima

at 364 nm derived from the differential spectra. (C) Quantiﬁcation of available free hemin (gray area of the graphs) in the presence of increasing concentrations of plasma

proteins. The quantiﬁcation analyses were based on the curves displayed on panel B. All measurements were performed in PBS pH 7.4. In all cases UV–vis absorbance spectra

of hemin were recorded in the wavelength range 300–700 nm in quartz optical cell with 1 cm path length. The ﬁgure shows representative results from two independent

experiments.

UV–vis absorbance of heme-hemopexin complex (Fig. 1C). Finally cysteine (0.156, 0.313, 0.625, 1.25, 2.5, 5, 10 and 20 mM) were mea-

we studied the most abounded heme binding protein in plasma sured. Fig. 2A shows differential absorbance spectra obtained after

concentration, which is albumin. The exposure of heme-albumin subtraction of the absorbance spectrum of hemin alone from the

complex to any of the studied low molecular weight heme ligands absorbance of hemin in the presence of different concentrations of

did not induce any considerable changes of the UV–vis absorbance cysteine. The spectroscopic change in differential spectra reached

spectrum (Fig. 1D). Collectively, these data suggested that among maximum at cysteine concentration of 2.5 mM. We decided to use

studied ligands of heme only cysteine did not affect the spectral somewhat higher concentration of 10 mM cysteine to ensure a bet-

properties of heme when in complex with the most pathologically ter sensitivity of the assay in a complex milieu of the human serum.

relevant heme-binding proteins. However, when cysteine inter- Next, we measured the UV–vis absorbance spectra of increasing

acts with free hemin there is an appearance of particular spectral concentrations of hemin (0, 0.625, 1.25, 2.5, 5, 10 and 40 ␮M)

changes, which are distinguishable from the changes occurring before or after exposure to 10 mM of cysteine. Fig. 2B displays

upon heme binding to the proteins themselves. Consequently, we differential absorbance spectra obtained after subtraction of the

considered cysteine as the most appropriate probe for developing absorbance of hemin alone from the absorbance spectrum of hemin

a strategy for detection and quantiﬁcation of free heme in complex in the presence of cysteine. The obtained data clearly demonstrated

systems such as blood plasma or serum. that the presence of cysteine resulted in a marked blue shift in

the absorbance at Soret region with maximum of 364 nm. The

absorbance intensity at 364 nm increased linearly with augmenta-

3.2. Titration experiments

tion of the hemin concentration (Fig. 2A). This allowed construction

of plot of hemin concentration versus absorbance increase at

To determine the appropriate concentration of cysteine to be

364 nm, which could be used for quantiﬁcation of protein-unbound

used as a probe for heme quantiﬁcation, the UV–vis absorbance

heme.

spectra of 20 ␮M hemin with the increasing concentrations of

316 R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319

3.3. Validation of the spectroscopy assay by use of different

heme-binding proteins

Further, we assessed the potential to use cysteine as a probe

for detection and quantiﬁcation of free heme in the presence of

typical plasma heme-binding proteins. Human hemopexin binds

−13

heme with extremely high afﬁnity (KD value of < 10 M) [6].

This protein circulates in plasma at approximate concentration of

1 mg/ml (17 ␮M). On the other hand human albumin binds heme

-8

with signiﬁcantly lower afﬁnity (KD value of 10 M) [6,15]. Despite

characterized by a relatively low afﬁnity, the interaction of heme

with albumin is physiologically relevant as this protein is present in

human plasma at high concentration - 35–50 mg/ml (520–750 ␮M).

Indeed, complexes of heme with albumin have been detected in

case of severe hemolytic conditions.

Besides, prototypic heme-binding plasma proteins, previous

studies have demonstrated that a substantial fraction of IgG in

healthy individuals is able to interact with heme [16]. Therefore, in

our analyses we also included pooled human IgG (pIgG) obtained

from blood of > 3000 healthy donors and as control a monoclonal

human IgG1 that was formerly identiﬁed as heme-binding anti-

body.

To evaluate heme-binding capacity of albumin, hemopexin and

IgG, a ﬁxed concentration of hemin (20 ␮M) was added to increas-

ing concentrations of the plasma proteins in absence or presence of

a high molar excess of cysteine (10 mM). The spectra of absorbance

of heme-protein complex at given concentration of protein were

subtracted from the corresponding spectra of heme-protein com-

plex in the presence of cysteine. The obtained differential spectra

are displayed on Fig. 3A. As can be observed a decrease of protein

concentrations resulted in an appearance and a progressive aug-

mentation of absorbance peak at 364 nm in the differential spectra,

indicating presence of free hemin. Notably, at high protein con-

centrations both differential absorbance spectra of albumin and

hemopexin displayed an additional peak of absorbance at 410 nm

in the presence of cysteine. This peak, however, is easily distin-

guishable and not interfering with the absorbance peak at 364 nm.

In order to quantify free hemin, the absorbance maxima at

364 nm typical for interaction of cysteine with free hemin were

plotted versus molar concentration of proteins (Fig. 3B). These

plots were further used to estimate the percentage of uncomplexed

hemin (Fig. 3C). One strategy for quantiﬁcation of free hemin is

to use calibration curve as this displayed on Fig. 2. Another strat-

egy consist in quantifying unbound hemin as proportion change

in absorbance at 364 nm compared to the maximal change of the

hemin in absence of protein (black lines in Fig. 3A).

The obtained data revealed that at 10 ␮M human albumin was

able to scavenge completely 20 ␮M of hemin. Although hemopexin

binds hemin with considerably higher afﬁnity, complete scaveng-

ing of detectable free heme was reached in the presence of 20 ␮M

(i.e. equimolar concentration) of hemopexin (Fig. 3C). This result

can be explained by the fact that albumin possesses more than one

binding site for heme. Indeed, early spectroscopy studies identi-

ﬁed the presence of second afﬁnity binding site for heme on the

Fig. 4. Evaluation of heme-binding capacity of human serum. Human serum from

human albumin [17]. In contrast, structural studies have indicated healthy donor was diluted in the range 20–5120 fold and exposed to 20 ␮M of hemin.

that hemopexin has only one binding site for heme [6,18]. Cysteine UV-absorbance spectra were recorded in the absence and in the presence of 10 mM

cysteine. The spectra of different dilutions of the human serum were also recorded

is able efﬁciently to distinguish protein-free from protein-bound

and used for correction of the turbidance effect. (A) Differential spectra obtained

heme. The quantiﬁcation of free heme in the presence of pooled

after subtraction of heme-serum spectrum at given dilution from the correspond-

human IgG, indicated that polyclonal antibodies had markedly

ing spectrum in the presence of excess of cysteine. The black line represents the

␮

lower heme-binding capacity. Indeed, KD value of ca. 5 M for heme differential spectra of hemin in the absence of serum. (B) Serum dilution-dependent

changes in absorbance maxima at 364 nm derived from the differential spectra. (C)

binding to pIgG was recently documented [19]. Consequently, we

Quantiﬁcation of available free hemin (gray area of the graphs) as a function of

used very high concentration of pIgG (320 ␮M). However, even this

increasing serum dilutions. The quantiﬁcation analyses were based on the curves

high protein concentration was not able to scavenge completely the

displayed on panel B. All measurements were performed in PBS pH 7.4. The UV–vis

␮

available (20 M) of hemin (Fig. 3C). This result is consistent with absorbance spectra were recorded in the wavelength range 300–700 nm in quartz

the observation that only a fraction of antibodies in healthy humans optical cell with 1 cm path length. The ﬁgure displays representative results from

two independent experiments.

are able to interact with heme [16]. Indeed, a monoclonal IgG1

R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 317

Fig. 5. Measurement of heme in serum from mice with acute intravascular hemolysis. Left panel depicts UV–vis absorbance spectra of 40 × diluted mouse serum in the

absence (blue line) or presence of 10 mM cysteine (red line). The serum was obtained 24 h after induction of intravascular hemolysis. Right panel shows differential spectra

after the subtraction of the UV–vis absorbance spectrum of hemolytic serum from hemolytic serum in the presence of cysteine. The UV–vis absorbance spectra were recorded

in the wavelength range 300–700 nm using quartz optical cells with 1 cm path length. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred

to the web version of this article).

identified in our laboratory as heme-binding antibody, efficiently significant quantities of heme and contribute to the remaining

scavenged available 20 ␮M of hemin at protein concentration of > 200 ␮M heme-binding capacity. However, it should be noted

10 ␮M. The capacity of the monoclonal antibody to bind to two- that heme can oxidize LDL and oxidized LDL is regarded as

molar excess of heme can be readily explained by the fact that IgG pro-atherogenic [20]. Moreover, when bound to certain hemo-

is bivalent with two identical antigen-binding sites. proteins oxidized heme can also retain pathogenic potential [21].

Cysteine and GSH are reducing agents that can reduce bisul- The question of which form(s) of heme are pathologically rel-

ﬁde bonds in proteins. To assess whether this process occurs in our evant in vivo remains still open in the literature. If only heme

setting, we assessed the effect of cysteine and GSH on integrity of bound to hemopexin is functionally quenched and cannot exert

human IgG molecule. Surface immobilized anti-human light chain pro-inﬂammatory and cytotoxic effects, then heme-scavenging

antibodies (anti-␬ chain and anti-␭ chain) were used to capture capacity of serum is rather low. However, much higher protective

native or the treated IgG, followed by detection of constant portion capacity of serum constituents can be anticipated if only protein

of heavy chain of immunoglobulins with another antibody spe- unbound heme is pathogenic. In these considerations one should

ciﬁc for human Fc. As can be observed on Supplementary Figure take into account that heme bound to albumin may not be recycled

S3 exposure of human IgG to GSH or cysteine did not result in loss rapidly since the albumin’s circulatory half-life is >20 days. This

of structural integrity of IgG molecule i.e. there is not dissociation implies that in cases of chronic intermittent hemolysis the heme-

of heavy and light immunoglobulin chains that usually occurs upon binding capacity of albumin might be decreased or completely

reduction of IgG molecule. saturated, regardless of transfer of part of heme from albumin to

hemopexin and further transfer to liver. In this respect, the heme

detection strategy proposed in the present article can be useful for

3.4. Evaluation of heme-binding capacity of normal serum

evaluation of the total heme-binding capacity of serum of patients

with different hemolytic diseases. We expect that as lower is the

In order to test the cysteine-based strategy for determination

heme-binding capacity of patients’ sera as the risk for pathogenic

of protein-unbound heme in more complex and physiologically

effects of protein free or loosely bound heme is elevated.

relevant system, we estimated the heme-binding capacity of nor-

Finally, to assess whether in condition of acute intravascular

mal human serum. The ability of serum diluted in the range from

hemolysis there is free extracellular heme, we analysed serum

20 – 5120 -folds to scavenge a ﬁxed concentration of hemin

from phenylhydrazine treated mice. Phenylhydrazine is a redox-

(20 ␮M) was evaluated as described above for the individual pro-

active substance that upon administration in vivo causes abrupt

teins. The obtained data indicated that the protein-unbound hemin

and extensive hemolysis. The use of cysteine as a spectroscopy

is detectable only following minimum 90-folds dilution of the

probe demonstrated that there is no any detectable free heme in

human serum (Fig. 4). Further dilution of the serum resulted in

mouse serum following acute intravascular hemolysis. This result

progressively increased amounts of unbound hemin. Since the

is in accordance with the estimated considerable heme-binding

concentration of externally added hemin used in these experi-

capacity for human serum. Nonetheless, one can speculate that in

ments is ﬁxed at 20 ␮M, it can be estimated that undiluted normal

conditions of chronic intermittent hemolysis, when heme-binding

human serum has potential to bind approximately 1.8 mM of

capacity of plasma is saturated, the free heme can be detected

heme. It can be deduced that albumin is responsible for the pre-

(Fig. 5).

dominant fraction of the heme-binding capacity of serum. Thus,

the available 520–750 ␮M of albumin would be able to buffer

1–1.5 mM of free heme. The contribution of the hemopexin to 3.5. Evaluation of heme-binding capacity of normal serum by

the heme-binding capacity of human serum is signiﬁcantly lower apo-HRP assay

– 17 ␮M. Nevertheless, this can be sufﬁcient to ensure transfer

of extracellular heme to the liver for ultimate degradation by Finally, we compared the proposed approach with a strategy

heme oxygenase-1 and recycling of apo-hemopexin. Other con- that has been used for detection of free intracellular heme. Recovery

stituents of serum such as immunoglobulins, lipoproteins (HDL, of the enzymatic activity of apo-horseradish peroxidase (apo-HRP)

LDL) and alpha-1-microglobulin together may also accommodate provides convenient way for assessment of protein-unbound heme

318 R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319

approaches for heme detection and quantiﬁcation. In recent years

a considerable success has been achieved in the design of intracel-

lular ﬂuorescent or enzymatic probes for detection of free heme.

These probes are recombinantly expressed, have high sensitivity

and selectivity for heme and have been applied successfully for

the quantiﬁcation of free heme and study of heme trafﬁcking in

pathogens and model organisms such as malaria parasites [22],

Caenorhabditis elegans [23], and Saccharomyces cerevisiae [24]. A

strategy has also been developed for measurement of hemoglobin-

unbound heme in healthy human erythrocytes. Some label-free

approaches for identiﬁcation of heme have been also proposed

[25,26].

Despite the efﬁcient employment of techniques for detection

of intracellular heme, the strategies for quantiﬁcation of heme in

plasma or serum are less advanced and they yield more conﬂict-

ing results. The ﬁrst-generation techniques relayed on colorimetric

assay or reconstruction of peroxidase activity by heme [27]. These

methods, however, cannot distinguish heme that is bound to

hemoglobin from heme-bound to other proteins or free heme and

are therefore appropriate only for assessment of total heme in

plasma. More speciﬁc are attempts that relay on use of immunoas-

says for detection of plasma hemoglobin. Again these assays are not

capable to estimate heme that is in complex with plasma proteins

or free heme. Other techniques relayed on fractionation of plasma

and estimation of heme concentrations in protein-free fraction [28].

Recently a methodology for estimation of heme in plasma has

been proposed that relay on deconvolution of data from UV–vis

absorbance spectroscopy [29]. The advantage of this assay is that

it can well distinguish oxidized and reduced forms of hemoglobin

from rest of plasma cell-free heme. A high concentration of non-

hemoglobin extracellular heme (up to 50 ␮M in some patients) was

detected by applying this technique to plasma samples from sickle

cell patients. However, this approach cannot speciﬁcally estimate

heme species that are not in complex with the plasma proteins.

Another recently proposed technique speciﬁcally detects free

Fig. 6. Evaluation of heme-binding capacity of human serum by apo-HRP assay.

heme by using heme-binding antibody fragments (scFv) [30]. The

Human serum from healthy donor was diluted in the range 10–2560 fold and

advantage of this technique is that it determines protein free

exposed to 10 ␮M of hemin in presence or absence of apo-HRP. (A) Peroxidase

heme or heme that binds to scFvs with higher afﬁnities than to

enzyme activity was assessed by oxidation of ABTS (absorbance readings at 414 nm).

The curve was generated by subtraction of the peroxidase activities of control sam- plasma proteins. The authors utilized the technique for evalua-

ples with separately added hemin and apo-HRP from identical serum dilutions

tion of heme-binding capacity of mouse plasma. Moreover, the

incubated in the presence of both hemin and apo-HRP (B) Quantiﬁcation of available

same study revealed that the induction of hemolysis in C57BL/6

free hemin (grey area of the graphs) as a function of increasing serum dilutions. The

mice by phenylhydrazine did not result in saturation of the heme-

quantiﬁcation analyses were based on the curves displayed on panel A. (For inter-

pretation of the references to colour in this ﬁgure legend, the reader is referred to binding capacity of mouse plasma. It was observed only a transient

the web version of this article). reduction of the heme-binding capacity of the plasma at 6 h fol-

lowing induction of the hemolysis. This study also demonstrated

that the heme-binding capacity of plasma was not saturated follow-

[11]. We applied this technique for measurement of free heme

ing hemolysis in mouse models of sickle cell disease and malaria

in human serum. The obtained data demonstrated that reconsti-

[30]. Nonetheless, by applying a cellular system with peroxidase

tution of peroxidase activity was achieved at considerably lower

reporter authors demonstrated that in the three hemolytic models

serum dilutions as compared with heme detected by cysteine. Thus,

there was a release of free heme in range 2–5 ␮M. It remains, how-

available heme was detected at serum dilution of 20× and at dilu-

ever, unclear whether reporter cells acquire directly free heme or

tion of 100× more than 90% of added heme (10 ␮M) was available

they ﬁrst interact with heme-protein complexes and then heme is

for reconstitution of the enzymatic activity of apo-HRP (Fig. 6).

transferred intracellularly.

This result could be explain by the fact that apo-HRP is character-

Here we proposed alternative strategy that speciﬁcally deter-

ized by high heme binding afﬁnity [11] and hence it can compete

mines protein free heme. The main advantage of this technique

with heme bound with low afﬁnity to serum proteins. This result

over previous approaches is that heme-detection does not depend

once again demonstrated the feasibility of using cysteine as probe

on the heme binding afﬁnity for hemoprotein or plasma proteins.

for detection of free heme with higher capacity to discriminate

Other advantages of the proposed method are that it is rapid and it

between protein-bund (irrespectively of afﬁnity) from free heme.

requires only simple equipment and reagents.

A shortcoming of the presented method is that it has relatively

3.6. General reﬂections and conclusion low sensitivity i.e. minimal reliable detection of heme is ca. 0.5 ␮M.

The low sensitivity may hamper the application of the strategy

The important functions of heme that is unbound to hemo- for direct and precise estimation of low free heme concentrations,

proteins in processes such as cell signalling, regulation of gene as for example of those normally found intracellularly. However,

expression and cell metabolism as well as its pathophysiologi- as previously reported quantities of total extracellular heme are

cal relevance, have demanded development of novel assays and often in micromolar range in cases of intravascular hemolysis, the

R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 319

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