Oxidative Damage of Lysozyme and Human Serum Albuminand Their Mixtures. A Comparison of Photosensitizedand Peroxyl Radical Promoted Processes

Andrea Arenas • Rodrigo Vasquez •

Camilo Lopez-Alarcon • Eduardo Lissi •

Eduardo Silva

Published online: 5 July 2011

� Springer Science+Business Media, LLC 2011

Abstract Oxidative modifications of lysozyme (Lyso)

and human serum albumin (HSA) mediated by photoin-

duced processes and peroxyl radicals were studied. Both

oxidative conditions were applied to the separate proteins

and their mixtures. Dimerization and fragmentation of the

proteins do not correlate with the formation of carbonyls or

peroxides, implying that evaluation of these changes is not

an index of the overall oxidative modification of a protein.

The results obtained also show that the hypothesis that the

electrostatic interactions of Lyso and HSA could facilitate

the formation of Lyso-HSA dimers in the presence of a

source of reactive oxygen species was verified in both

ROS-producing systems.

Keywords Oxidative damage � Lysozyme � Human

serum albumin � Peroxyl radicals � Photosensitized

processes � Reactive oxygen species

Abbreviations

HSA Human serum albumin

Lyso Lysozyme

RF Riboflavin

MB Methylene blue

AAPH 2,20-azobis(2-amidinopropane) dihydrochloride

1 Introduction

The reactions of Reactive Oxygen Species (ROS) with

proteins produce reversible and/or irreversible modifica-

tions that lead to a progressive loss of the protein function.

These changes result from modifications of the protein

primary structure, with concomitant changes in the sec-

ondary and/or tertiary protein conformation. In addition,

these reactions could induce the irreversible fragmentation,

dimerization and/or oligomerization of the macromolecule.

The relevance of the latter processes depends on the

characteristics of the protein, the type of ROS, and the

interactions between the proteins present in the system.

Particularly important among the ROS implicated in the

oxidative damage of proteins, are free radicals such as

peroxyl radical and photochemically generated species

such as singlet oxygen. Peroxyl radicals react efficiently

with amino acids such as cysteine (Cys), tryptophan (Trp)

and tyrosine (Tyr), generating Cys dimers, Trp metabolites

and di-tyrosine adducts, respectively [12]. Photochemical

processes are usually mediated by photosensitizers such as

riboflavin (RF) and methylene blue (MB). RF, commonly

known as vitamin B2, is an endogenous photosensitizer

that induces photomodifications by Type-I and Type-II

mechanisms, while MB initiates the oxidative modification

of biomolecules mostly through a Type-II mechanism

[3, 19, 25, 26, 29]. In the case of RF, the mechanism

involves the generation of a short-lived (ca. 12 ns) singlet

state (1RF) that converts to the triplet state (3RF) with a

A. Arenas � R. Vasquez � E. Silva (&)

Departamento de Quımica Fısica, Facultad de Quımica,

Pontificia Universidad Catolica de Chile, C.P. 782 0436

Santiago, Chile

e-mail: esilva@uc.cl

C. Lopez-Alarcon

Departamento de Farmacia, Facultad de Quımica, Pontificia

Universidad Catolica de Chile, Santiago, Chile

E. Lissi

Facultad de Quımica y Biologıa, Universidad de Santiago de

Chile, Santiago, Chile

123

Protein J (2011) 30:359–365

DOI 10.1007/s10930-011-9341-1

high intersystem crossing quantum yield (UISC = 0.67).

Once formed, 3RF can react by both Type I and Type II

mechanisms. Hydrogen atom or electron transfer processes

involving 3RF can produce free radicals or radical ions (or

both) and subsequent reactions of the flavin or protein

centered radicals can generate hydrogen peroxide (H2O2),

hydroxyl radicals (.OH) and superoxide anion radical (O22)

[19, 26]. In the Type II process, energy transfer from the

excited triplet to a ground state oxygen molecule generates

singlet oxygen (1O2), which can also modify proteins,

probably by reacting primarily with Cys, Trp and Tyr

residues.

The oxidative damage of proteins triggered by ROS

could favour the occurrence of covalent binding between

amino acids present in the interacting macromolecules

(covalent dimerization and/or covalent oligomerization

processes). In the presence of protein mixtures, these

considerations would apply to covalent dimerizations tak-

ing place between the same or different species. In spite of

the fact that these irreversible protein-protein cross-lin-

kings could lead to aggregation of proteins in a variety of

physiological processes, such as in the formation of cata-

racts, the efficiency of ROS-mediated protein dimerization/

oligomerization has been relatively little studied (in par-

ticular when the process takes place between different

proteins). Furthermore, very little is known regarding how

the efficiency of the process is modulated by pre-associa-

tion of the macromolecules and/or by the type and locali-

zation of the ROS source.

Lysozyme (Lyso) and human serum albumin (HSA),

two well characterized water-soluble globular proteins,

constitute good models to study the role that ROS may play

in the oxidative modification of biomolecules. Some

reports have shown that the interaction of ROS (chemically

or photochemically generated) with Lyso leads to protein

radicals and to a decrease in its catalytic activity [16, 18,

30]. Interestingly, these processes are efficiently inhibited

by melatonin, coumarins and other free radical scavengers

[16, 18, 22, 30]. Regarding HSA, it has been demonstrated

that it initially reacts with ROS at Cys-34 [12]. In addition,

HSA disulfide dimers have been generated in human

plasma in the presence of tert-butyl hydroperoxide [21].

The extremely different isoelectric point values of Lyso

(10.9) and HSA (4.9) favors aggregation to form biocon-

jugates between positively charged Lyso and negatively

charged HSA [6, 10]. This interaction offers the possibility

of testing the influence of the intermolecular interactions

on the susceptibility to covalent cross-linking, as observed

in tissues with supramolecular structures, such as the skin

and the eye lens.

In the present work, we evaluate the oxidative damage

to Lyso and HSA induced by hydrophilic peroxyl radicals

and photochemical processes. AAPH (2,20-azobis-

(2-amidinopropane)) was used as the peroxyl radical

source, while RF and MB were used as photosensitizers to

generate reactive species. We hypothesized that the gen-

eration of protein free radicals would lead to dimers and

oligomers, a process that could be favored by non-covalent

pre-aggregation of the macromolecules.

2 Materials and Methods

Human serum albumin (HSA) and lysozyme from chicken

egg white (Lyso), riboflavin (RF), Methylene blue (MB),

Xylenol orange (XO), 2,20-azobis(2-amidinopropane)

dihydrochloride (AAPH), ferrous ammonium sulfate, tri-

chloroacetic acid (TCA), and 2,4-dinitrophenylhydrazine

(DNPH) were purchased from Sigma.

2.1 Protein Oxidation by Peroxyl Radicals

The oxidation of proteins was promoted by peroxyl radi-

cals generated in the thermal decomposition of AAPH in

aqueous solution (100 mM phosphate buffer, pH 7.4). For

this purpose, the proteins (3 mg/mL) were incubated in the

presence of AAPH (10 mM) at 37 �C in air saturated

solutions.

2.2 Irradiation Conditions

Solutions of proteins (0.33 mg/mL) and MB or RF

(35 lM) were irradiated at 37 �C in a Perspex water-

jacketed cell (wall thickness 4.0 mm; internal path length

1 cm) illuminated by a 150 W, 24 V Osram Bellaphot

Halogen lamp (Germany) from a slide projector. During

the irradiation, the solutions were gently bubbled with air.

The commercial Tungsten-halogen lamp used in this work

generates a continuous distribution of light across the vis-

ible region, with a very weak emission in the ultraviolet

portion of the spectrum. The 4.0 mm wall thickness of the

Perspex cell, together with the ordinary glass and plastic

components of the slide projector optical system, filter out

all the light below 320 nm. The slide projector cooling fan

limits the temperature of the halogen lamp bulb, with a

concomitant decrease in light emission below 400 nm.

The quantum yields of Trp photodecomposition in the

presence of MB (U = 0.0094) and RF (U = 0.023 [7]

were used as actinometers for the determination of the

quantum yields reported in this work.

2.3 SDS–PAGE Analysis

Protein crosslinking and fragmentations were determined

by SDS–PAGE electrophoresis. Samples of pre-oxidized

protein solutions were boiled for 5 min in a 62.5 mM Tris

360 A. Arenas et al.

123

buffer (pH 6.8) solution containing 2% sodium dodecyl

sulfate (SDS), 10% glycerol, 100 mM of b-mercap-

toethanol (as reducing agent) and traces of bromophenol

blue (as a tracking dye). Acrylamide (3%) stacking gel, 12

and 8% acrylamide resolving gels and a running buffer

containing 25 mM Tris, 400 mM Gly and 0.1% SDS, pH

8.3, were used. Electrophoresis was performed at 100 V

during 1–2 h. Gels were stained with 0.1% Coomassie

Brilliant Blue and destained in a solution of methanol and

acetic acid during 48 h. Gels were scanned and the quan-

tification of crosslinked and fragmented protein was per-

formed by employing ImageJ software.

2.4 Carbonyl Residue Assay

Carbonyl residues in pre-oxidized proteins were deter-

mined according to the method described by Levine et al.

[15]. Briefly, aliquots of 330 lL of oxidized protein solu-

tions were incubated with 1.6 mL of 0.2% DNPH (pre-

pared in 2N HCl) for 30 min. Proteins were precipitated

with ice-cold TCA (to a final concentration of 20%) and

centrifuged (1,970 9 g) for 10 min. The resultant pellets

were washed three times with a solution of ethanol:ethyl

acetate (1:1) and dissolved in 6 M guanidine for 10 min at

37 �C. The absorbance was measured at 370 nm and the

carbonyl group concentration was estimated using an

extinction coefficient of 22,000 M-1 cm-1 [15].

2.5 Peroxide Assay

Total peroxides were quantified by Fox’s assay. Briefly,

420 lL of protein solution were mixed with 2 mL of the

Fox reagent (a mixture of 0.25 mM ferrous ammonium

sulfate and 125 lM Xylenol orange (XO) in 25 mM sul-

furic acid). After 30 min incubation, the absorbance was

determined at 560 nm. The concentration of peroxides was

estimated from the absorbance employing a calibration

curve obtained using a commercial solution of hydrogen

peroxide. The amount of organic peroxides was estimated

by pre-incubating the protein solutions with 20 lL of

catalase (CAT, 715 U/mL) for 15 min prior to the addition

of Fox’s reagent (2 mL).

3 Results and Discussion

3.1 Oxidative Modification of Single Proteins

We have evaluated the damage inflicted on two globular

proteins, lysozyme (Lyso) and human serum albumin

(HSA), by peroxyl radicals generated by the aerobic ther-

molysis of AAPH and by singlet oxygen or radical species

generated by Methylene blue (MB) or riboflavin (RF)

photosensitization. After exposure of the proteins to these

oxidative processes, we measured the formation of car-

bonyl groups, peroxides and the oligomerization and

fragmentation of the proteins. Figure 1 shows representa-

tive electrophoretic patterns of Lyso and HSA solutions

exposed to RF-sensitized photooxidation. The simulta-

neous occurrence of protein dimerization and fractionation

is clearly indicated. In order to quantify the efficiency of

the observed modifications, the gels were submitted to

densitometric analysis. The results were expressed as either

quantum yields (for the photosensitization reactions) or in

terms of the damage inflicted per peroxyl radical formed in

the system and are collected in Table 1. The results indi-

cate that, for both proteins, the major deleterious effect was

produced by AAPH-derived peroxyl radicals. This inter-

action of peroxyl radicals with Lyso and HSA induces

oxygen mediated oxidative modifications, cross-linking

reactions and fragmentation, the net damage being greater

for Lyso than for HSA. The data in Table 1 imply that

peroxyl radical production and Lyso modification have

similar values. Thus, even though the yield of peroxy

groups produced in the protein might be overestimated due

to contribution of peroxyl radical-derived peroxides, this

suggests that there is almost quantitative trapping of the

azo-derived radicals by the enzyme [16], with an important

fraction of these trapping events leading to protein cleav-

age or irreversible oligomerization.

kDa100.0

75.0

50.0

37.0

25.0

20,0

15.0

10.0Lyso

Lysodimer

Lyso

150.0100.075.0

50.0

37.0

25.0

HSA

HSAfragmentation

fragmentation

HSAdimer

B

Lya b c d e f g a b c d e f g

AFig. 1 SDS–PAGE of

lysozyme A, Lyso: 12%

acrylamide) and human serum

albumin B, HSA: 8%

acrylamide) exposed to visible

light in the presence of

riboflavin (RF). Lanes a–gcorrespond to MW standard, 0,

15, 30, 45, 60 and 75 min of

irradiation, respectively

Oxidative Damage of Lysozyme 361

123

The efficient modification of Lyso by AAPH-derived

radicals is somewhat surprising given the like (positive)

charge of the radicals and the protein. The high efficiency

of Lyso modification could reflect an amino acid compo-

sition with more residues that are prone to be easily oxi-

dized and amenable to crosslinking or fragmentation. Lyso

and HSA contain six and one Trp residues in their primary

structure, respectively. In particular, two of the six Trp

residues are on the surface of Lyso and are easily oxidiz-

able by peroxyl radicals [16, 17, 29]. On the other hand,

HSA possess many more Tyr residues (18) than Lyso (3).

Tyr residues have been associated with crosslinking reac-

tions through the formation of di-Tyr linkages [11] and

have also been identified in HSA adducts [14]. However,

the reactivity of tyrosyl residues is much lower than that of

other reactive residues, such as Trp or Cys [17].

Photosensitized protein modifications are more efficient

when RF is used as the sensitizer and Lyso is more sen-

sitive to photochemical modification than HSA. In both

systems (Lyso and HSA), dimeric compounds and aggre-

gates are present, together with carbonyl and peroxide

groups formed in the proteins by oxygen-mediated oxida-

tive processes. The data in Table 1 show that the relative

importance of the damages that are elicited depends

strongly on the protein and on the ROS source employed.

This implies that is not possible to assess the overall level

of oxidation of a protein from a single parameter, such as

the amount of carbonyl or peroxide groups introduced by

the oxidative process. This is particularly relevant since the

amount of carbonyls in proteins is frequently employed to

assess the degree of the oxidation. Indeed, the data in

Table 1 show that the amount of carbonyl groups is a very

poor predictor of the other damage inflicted on the protein

ensemble. This is further emphasized by the data presented

in Table 2, in which the modification quantum yields are

normalized relative to the carbonyl production. These data

show that the observed changes are strongly dependent of

the type of oxidative stress and the protein considered.

However, some generalizations can be established. In

particular, it can be concluded that dimerization and frag-

mentation are particularly inefficient for HSA and that the

effect is more evident when the oxidation is elicited by

exposure to AAPH-derived radicals. This could be related

to a dominant role of Trp modifications in these processes.

3.2 Oxidative Modification of Lyso/HSA Mixtures

HSA and Lyso possess acidic (pI = 4.9) and basic

(pI = 10.9) isoelectric points, respectively. Thus, consid-

ering their respective characters of poly-anion and poly-

cation at pH 7.4, the attractive electrostatic interaction

should favor non-covalent association of HSA and Lyso in

solutions containing both proteins [10]. In fact, it has been

observed by ultracentrifugation that Lyso binds to HSA

with a binding constant of ca. 2 9 104 M-1 at pH 7.4 [5].

This implies that, under our working conditions, most of

the HSA and more than 30% of the Lyso are involved in

non-covalent aggregates. On the other hand, more drastic

conditions are required to generate significant amounts of

HSA or Lyso self-aggregates [20, 23].

Figure 2 shows the electrophoretic patterns of a solution

containing both HSA and Lyso after exposure to visible

light in the presence of RF. As can be seen in this figure,

simultaneously with the generation of Lyso-Lyso and

HSA-HSA dimers, there is also intermolecular Lyso-HSA

crosslinking, characterized by the clear presence of a new

band at ca. 75 kDa. The results obtained for dimer for-

mation in the oxidation of Lyso/HSA mixtures are col-

lected in Table 3. These data indicate that self-dimerization

of Lyso is always more important than dimerization of

HSA. This result is similar to that obtained employing the

separate proteins (Table 1), and may reflect a dominant

role of Trp groups in the irreversible dimerization associ-

ated with the protein oxidation. However, other factors

might contribute to the reduced dimerization quantum

yields for HSA. In particular, the larger size of the HSA

Table 1 Quantum yields of protein modifications elicited by peroxyl

radicals or following irradiation of riboflavine or methylene blue

ROS

source

Udimer

9 103

Ufragmentation

9 103

Ucarbonyls

9 103

Uperoxides

9 103

Lyso

RF 7.8 ± 0.2 1.7 ± 0.6 0.36 ± 0.10 0.28 ± 0.05

MB 0.55 ± 0.81 1.7 ± 0.1 1.6 ± 0.4 0.047 ± 0.008

AAPHa 140 ± 40 300 ± 10 130 ± 40 350 ± 50

HSA

RF 0.40 ± 0.03 0.90 ± 0.01 3.0 ± 0.2 0.20 ± 0.09

MB 0.10 ± 0.09 0.22 ± 0.09 0.32 ± 0.02 0.062 ± 0.028

AAPHa 21 ± 8 10 ± 4 170 ± 11 280 ± 64

a These values represent the rate of protein modification/rate of peroxyl

radicals production

Table 2 Modification quantum yields normalized by carbonyl

groups yields

System Udimer/

Ucarbonyl

Ufragmentation/

Ucarbonyl

Uperoxide/

Ucarbonyl

RF/Lyso 22 ± 6 4.7 ± 0.8 0.8 ± 0.1

MB/Lyso 0.34 ± 0.09 1.1 ± 0.2 0.03 ± 0.01

AAPH/Lysoa 1.1 ± 0.3 2.3 ± 0.4 2.7 ± 0.4

RF/HSA 0.13 ± 0.02 0.30 ± 0.05 0.07 ± 0.01

MB/HSA 0.31 ± 0.08 0.7 ± 0.1 0.20 ± 0.03

AAPH/HSAa 0.12 ± 0.03 0.06 ± 0.01 1.8 ± 0.3

a These values represent the rate of protein modification/rate of

peroxyl radicals production

362 A. Arenas et al.

123

molecule could disfavour the occurrence of specific inter-

protein contacts by increasing the proportion of non-reac-

tive ‘‘deeply buried’’ radicals. Furthermore, for surface

located radicals, the large size of HSA could decrease the

rate of their diffusion-controlled bimolecular combination.

The expectation that formation of irreversible Lyso/

HSA adducts would be favoured by the reversible pre-

association of the proteins due to their electrostatic

attraction is qualitatively supported when the damaging

species are the hydrophilic peroxyl radicals or the radical

intermediaries generated in RF sensitized photo-processes

(Table 3). However, it has to be taken into account that the

factors limiting the formation of Lyso/HSA adducts are

different depending on the oxidative source. Thus, the

initial radicals reach the proteins one by one in the AAPH-

induced process, while in the RF-sensitized process two

radicals are formed simultaneously in the adduct.

In the AAPH system, once a free radical is generated in

a protein/protein adduct, the mean time required to produce

a second radical in the partner protein (tpair) can be esti-

mated as

tpair ¼ ½protein�=Rate of radical production ð1Þ

Under our experimental conditions, this amounts to ca.

60 min, a time that can be considered to be a lower limit

since it assumes that all radicals react with the protein and

that covalent binding occurs quantitatively when a radical

is formed in each partner of the adduct. This simple

calculation allows us to conclude that covalent bond

formation requiring the simultaneous presence of a pair of

radicals in the adduct cannot be an operative pathway, at

least at the low rates of radical production employed in the

present work. Protein dimer formation must then result

from a random walk of two protein radicals. The steady-

state concentration of protein radicals will depend upon the

rates of radical formation (0.8 lM/min) and removal. If the

radical combination rate constant is assumed to be ca.

106 M-1 s-1, a typical value for the termination step in

free radical-mediated polymerizations, the steady-state

concentration of protein radicals would be ca. 1.1 9

10-7 M, leading to a life expectancy of ca. 9 s. Thus,

radical combination resulting from the random walk of

single protein radicals is a much more likely mechanism

for dimer formation than covalent binding of pre-formed

radical pairs.

If random walk encounters are the dominant pathway for

dimer formation, the yields of dimers are related to the

corresponding bimolecular rate constants by the following

equation.

ðkLyso=HSAÞ2=kLyso=Lyso kHSA=HSA

¼ ðULyso=HSAÞ2=ULyso=LysoUHSA=HSA ¼ 4:0 ð2Þ

The above discussion assumes that the dimerization

involves only the primary protein radicals and disregards

the occurrence of secondary processes such as reaction of

carbonyl groups with protein amine groups, which could

also lead to covalent protein–protein associations. The

relevance of these secondary inter-protein processes can be

ascertained from the time profile of aggregate

accumulation. In particular, secondary reactions should

produce clear upward curvature in the dimer versus time

plots. The data depicted in Fig. 3 for Lyso dimers argue

against a significant contribution of secondary processes to

aggregate formation.

The photosensitized processes possess several funda-

mental differences compared to the AAPH-promoted

modifications. In particular, non-radical processes and the

formation of radical pairs can take place inside the protein

kDa

150.0

100.0

75.0

50.0

37.0

25.0

HSA-Lyso

Lyso

Lysodimer

HSA

a b c d e f g

HSAdimer

Fig. 2 SDS–PAGE of a mixture of lysozyme (Lyso) and human

serum albumin (HSA) exposed to visible light in the presence of

riboflavin (RF). Lanes a–g correspond to MW standard, 0, 15, 30, 45,

60 and 75 min of irradiation, respectively

Table 3 Quantum yields of dimers formation for the oxidation of

Lyso/HSA mixtures

ROS

source

Lyso/

Lyso 9 103Lyso/

HSA 9 103HSA/

HSA 9 103

RF 0.9 ± 0.1 2.3 ± 0.6 0.040 ± 0.005

MB 0.69 ± 0.02 0.21 ± 0.03 0.036 ± 0.014

AAPHa 2.0 ± 0.3 40 ± 6 \0.2

a These values represent the rate of protein dimerization rate/rate of

peroxyl radicals production

Oxidative Damage of Lysozyme 363

123

and/or protein complexes when the dyes are bound to

individual proteins or their aggregates. Indeed, at the pro-

tein concentrations employed in the present work, most of

the dye is bound to the proteins, in particular to HSA. Thus,

the binding constant of MB to HSA of 1.5 105 M-1 [1]

predicts that 90% of the dye is bound to to the protein at

50 lM HSA. A similar efficiency of binding to HSA has

been reported for RF [13]. Another important difference is

that the interaction of the excited dye with the protein

greatly favors the Type I process, which always generates

radical pairs.

The quantum yield for Lyso-Lyso dimer formation in

the presence of RF is significantly higher than that

observed for dimerization of HSA using the same photo-

sensitizer. This is in agreement with the report that the

quenching of the flavin triplet state by BSA, monitored by

laser flash photolysis, is less efficient than that by Lyso

[28]. The proposed reaction is electron transfer from the

Trp moiety to the flavin triplet [26], which gives rise to

radical intermediates responsible of protein crosslinking

[27]. The quenching of 3RF by HSA should be less efficient

than by BSA considering that the former contains only one

Trp residue compared to two and six residues in BSA and

Lyso, respectively. In addition, the generation of di-Tyr,

which also is a source of protein crosslinking, has been

observed when this amino acid is irradiated using RF as

sensitizer [24]. The Tyr-mediated dimerization of hen and

turkey egg-white Lyso has been observed after oxidation

by .OH or N3. free radicals [2]. Like 3RF, N3

. reacts pref-

erentially with tryptophan residues. Tyr can be oxidized by

long-range intramolecular electron migration [4, 9]. The

hydroxyl radical,.OH, which is also formed in the RF-

sensitized oxidation of proteins [8], may react directly with

tyrosines, with the accessibility to solvent of the Tyr resi-

due playing an important role in di-Tyr formation.

The efficiency of RF sensitized carbonyl production is

ten times higher in HSA than in Lyso, which can be

attributed to an increased contribution of the Type II RF

sensitized mechanism in the case of HSA, reflecting the

less efficient 3RF quenching by this protein.

Cross-combination between proteins is particularly

favored when RF or AAPH are employed as sensitizers. In

fact, the data of Tables 3 show that the cross-combination

ratio ðULyso=HSAÞ2=ULyso=LysoUHSA=HSA is ca. 150 for RF

and [4,000 for AAPH. This predominance of cross-com-

bination can result from a conbination of several factors,

such as HSA/Lyso association favoured by electrostatic

interactions and a predominance of a Type I mechanism

promoted by RF binding to the proteins [13, 28]. A Type I

pathway would produce two radicals simultaneously, a

situation that could promote formation of covalent links

between preformed protein pairs. As mentioned above,

collision between radical-bearing proteins is the main

pathway for AAPH-mediated crosslinking. However, the

rate of cross-combination encounters could be favoured by

the polyanion and polycation character of Lyso and HSA,

respectively.

In the case of MB, which is predominantly a Type II

sensitizer, the quantum yields of protein modification are

low for both proteins (0.0044 and 0.0008 for Lyso and

HSA, respectively). It is interesting to note that the effi-

ciency of albumin modification is low in spite of a sig-

nificant association of the dye to the protein [1]. This could

be due to the external localization of the dye at specific

positions dictated by electrostatic interactions between the

anionic protein and the cationic dye MB [1].

The exposure of a mixture of HSA and Lyso to visible

light in the presence of MB or RF leads to the formation of

HSA-Lyso, HSA-HSA, and Lyso-Lyso dimers. For both

sensitizers, the efficiency of HSA-HSA dimer formation in

the mixture is lower than that reported in Table 1 for the

separate proteins. In the presence of RF, HSA-Lyso dimer

formation takes place with a high quantum yield; simul-

taneously, a significant decrease in the generation of Lyso-

Lyso dimers is observed. The sum of the quantum yields of

all dimerization processes that occur in the protein mixture

(ULyso-Lyso ? UHSA-Lyso ? UHSA-

HSA = 3.2 9 10-3) is smaller than that observed for Lyso

alone (ULyso-Lyso = 7.8 9 10-3). Thus, despite the fact

that the electrostatic interaction between HSA and Lyso

favours non-covalent binding between these two proteins,

the total dimerization capacity is diminished, probably due

to a different distribution of the sensitizer. Irradiations

0 30 60 90 120 150 1800

2

4

6

8

10

12L

yso

-Lys

o c

on

cen

trat

ion

/ µ

M

Incubation time / min

Fig. 3 Dependence of the Lyso-Lyso formation with the incubation

time in presence of AAPH. Lyso (3 mg/mL) was incubated with

AAPH (10 mM) in aqueous solution (phosphate buffer 100 mM) at

pH 7.4. Aliquots were taken between 0 and 3 h and analyzed by SDS–

PAGE according to Materials and Methods section. p \ 0.0001,

n = 3

364 A. Arenas et al.

123

performed in the presence of MB also show the formation

of HSA-Lyso dimers, but the efficiency of this process

(UHSA-Lyso = 2.1 9 10-4) is lower than that for for-

mation of Lyso-Lyso dimers (ULyso-Lyso = 6.9 9 10-4)

in the same reaction mixture. In the MB-sensitized reaction,

the sum of the quantum yields of all dimerization processes

that occur in the protein mixture (ULyso-Lyso ? UHSA-

Lyso ? UHSA-HSA = 9.4 9 10-4) is larger (i.e., more

efficient) than that observed for Lyso alone (ULyso-

Lyso = 5.5 9 10-4), which suggests that the electrostatic

interactions between the proteins and between the cationic

sensitizer and the anionic Lyso improve the yields of the

dimerization processes.

4 Conclusions

The radical-induced fragmentation and dimerization of

HSA and Lyso do not correlate with the formation of

carbonyls and peroxides, implying that evaluation of these

latter changes is not a reliable index of the overall oxida-

tive modifications of a protein.

In the case of the Lyso/HSA mixture, the hypothesis that

the electrostatic interaction between Lyso and HSA could

promote the formation of Lyso-HSA dimers in the presence

of an oxidative source was verified when either peroxyl

radicals or a Type-I photosensitizer (Riboflavin) were

employed as the oxidative source.

Acknowledgments This work was supported by Proyecto Puente

05/2009 and FONDECYT (Grant 1070285).

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