Light-induced inhibition of papain by a {Mn-NO}6 nitrosyl: Identification of papain-SNO adduct by mass spectrometry

Article abstract of DOI:10.1016/j.jinorgbio.2005.04.002

Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S-NO bond formation a

Full text of DOI:10.1016/j.jinorgbio.2005.04.002

JOURNAL OF
Inorganic
Biochemistry
Journal of Inorganic Biochemistry 99 (2005) 1458–1464
www.elsevier.com/locate/jinorgbio
6
Light-induced inhibition of papain by a {Mn–NO}
nitrosyl: Identification of papain–SNO adduct by mass spectrometry
Raman K. Afshar, Apurba K. Patra, Pradip K. Mascharak *
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, United States
Received 8 February 2005; received in revised form 24 March 2005; accepted 31 March 2005
Available online 31 May 2005
Abstract
Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous
studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S–NO bond formation at the
active site while NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP), N-nitrosoaniline derivatives, and S-nitroso-glutathi-
one (GSNO) inhibit the enzyme via S-thiolation by thiyl radicals generated from the S-nitrosothiols. In this study, we report papain
6
ꢀ
inactivation by a photosensitive {Mn–NO} nitrosyl [(PaPy
N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide. This nitrosyl releases NO upon exposure to visible light of low
intensity (50 W tungsten lamp). With N -benzoyl-L-arginine-p-nitroanilide (L-BApNA) as the substrate, the dissociation constant for
the breakdown of the enzyme–inactivator complex (K ) and the overall inactivation rate constant (k ) were calculated to be 2.46 mM
3 4
)Mn(NO)](ClO ) (1) where PaPy3 is the anion of the designed ligand
a
I
i
ꢀ1
and 64.8 min , respectively. The papainS–NO adduct has been identified using electrospray mass spectrometry (ESI-MS). The
results demonstrate that controlled inactivation of papain can be achieved with the {Mn–NO} nitrosyl 1 and light. The reaction
6
is clean and the extent of inactivation is directly proportional to the exposure time.
ꢀ
2005 Elsevier Inc. All rights reserved.
Keywords: Manganese nitrosyl; Papain; Inhibition; NO donor; Mass spectrometry; Nitric Oxide; Light-induced inhibition
1
. Introduction
or nitrosylation [12]. Nitrosylation, a reversible ubiqui-
tous modification that is key to cellular signaling, targets
either metal–heme centers or thiol (–SH) residues in pro-
teins [13]. NO covalently modifies cysteine residues in
proteins via S-nitrosylation (–SNO), a reaction that
either activates or blocks a signaling pathway [14].
During the past few years, an increasing number of
intracellular and extracellular proteins have been shown
to be S-nitrosylated in vivo [1,14,15]. S-nitrosylation of
thiol (Cys) group(s) in proteins occurs in serum albumin
[16–18], mitochondrial complex I [19], tissue transgluta-
minase [20], cathepsin K [21], glyceraldehyde 3-phos-
phate dehydrogenase [22], ryanodine receptor [23],
protein kinase C [24], tissue-type plasminogen activator
[25], transcriptional activators [26], human immunodefi-
ciency virus protease [27], and caspases [28]. These pro-
teins are involved in numerous biochemical processes
Nitric oxide (NO), a highly reactive gaseous molecule
produced by the intracellular enzyme nitric oxide syn-
thase (NOS), participates in a variety of signaling path-
ways [1]. NO has been implicated as a key mediator in
physiological processes such as regulation of blood pres-
sure, modulation of neurotransmission, inhibition of
platelet aggregation, cell-mediated immune response,
antimicrobial activity, cellular respiration, and apopto-
sis [2–11]. Recent studies have shown that NO and re-
lated species regulate these diverse biological processes
by directly modifying proteins via oxidation, nitration,
*
Corresponding author. Tel.: +1 831 459 4251; fax: +1 831 459
2935.
E-mail address: pradip@chemistry.ucsc.edu (P.K. Mascharak).
0
162-0134/$ - see front matter ꢀ 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2005.04.002

R.K. Afshar et al. / Journal of Inorganic Biochemistry 99 (2005) 1458–1464
1459
2
+
including glycolysis, cell growth, Ca
homeostasis,
cial (and efficient) inhibitor of the cysteine protease
papain that can be conveniently controlled with light.
bronchodilation in human airways, vasorelaxation of
blood vessels, regulation of blood flow, immune re-
sponse, and apoptosis. Quite expectedly, the S-nitrosyla-
tion reactions have been looked into more closely in
relation to drug discovery. For example, the inactivation
of cathepsins, cysteine proteases involved in the inflam-
mation pathway, could lead to possible drug targets to
treat rheumatoid arthritis [29]. Cysteine proteases such
as vivapains are also involved in the parasitic hydrolysis
of hemoglobin and parasite growth [30]. The develop-
ment of inhibitors to these hemoglobinases could lead
to drug candidates against malaria.
O
NO
N
H
N
N
N
(
ClO4)
N
Mn
N
N
N
N
N
O
PaPy H
Complex 1
3
Papain, a 212 amino acid cysteine protease, is a plant
protease found in papaya. The active site of papain con-
tains the catalytic triad Cys25–His159–Asn175, which is
required for proteolysis [29,31]. The thiolate anion in
Cys25 serves as the nucleophile that attacks the carbonyl
function of the substrate to give an acyl enzyme interme-
diate and free amine. A water molecule then reacts with
the intermediate to give the carboxylic acid and the free
enzyme. Papain has a preference for bulky residues at
2. Experimental
2.1. Materials and methods
Papain, N -benzoyl-L-arginine-p-nitroanilide (L-BAp-
a
NA), dithiothreitol (DTT), acetonitrile (MeCN), ethyl-
enediaminetetraacetic acid (EDTA), glutathione
(GSH), hydrochloric acid (HCl), S-nitroso-glutathione
(GSNO), sodium chloride (NaCl), and potassium
biphosphate (KH PO ) were purchased from Sigma
the S subsite and small hydrophobic residues at the
2
S1 subsite [31]. Previous studies have shown that modi-
fication of the thiolate anion by alkylating or nitrosylat-
ing agents renders papain inactive [29,31]. Since caspases
and cathepsins are quite similar to papain, researchers
so far have utilized papain as the target in develop-
ment of effective inhibitors for rheumatoid arthritis
and malaria [31].
In a recent study, Wang and coworkers [32] have
shown that papain can be nitrosylated by a variety of
NO donors including glucose-S-nitroso-N-acetylpenicil-
lamine (glucose-SNAP-2) and S-nitroso-glutathione
2
4
Aldrich and used without further purification unless
otherwise noted. [(PaPy )Mn(NO)](ClO ) and [(PaPy )-
3
4
3
Mn(Cl)](ClO ) were synthesized by following a previ-
4
ously published procedure [37]. A Perkin Elmer
Lambda 40 spectrophotometer was used to record the
absorption spectra of the nitrosyls and the Papain–
SNO complex. It was also used in the kinetic studies.
High-pressure liquid chromatography (HPLC) analyses
were performed with a Spectra-Physics UV 2000 setup.
Mass spectra were recorded on a Waters ZMD Micro-
mass ESI Mass Spectrometer.
(
GSNO). The decomposition of the thiol-containing
NO donors releases NO, which in turn inactivates pa-
pain by nitrosylation of Cys25. The papain–SNO inter-
mediate eventually affords the S-thiolated species
papainS–SR as the final product. During the past two
years, we have been involved in the syntheses of metal
nitrosyls that photorelease NO upon illumination with
light (visible or UV) of low intensity [33–38]. In one such
system, nitrosyls derived from the Fe [33,34], Ru [35],
and Mn [37] complex of the designed ligand N,N-
bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carbox-
2.2. Stability of the [(PaPy )Mn(NO)](ClO ) complex
3
4
in buffer
The absorption spectrum of a freshly prepared solu-
tion of 1 in a 50 mM phosphate buffer (pH 6.2, 1 mM
EDTA, and 0.1 M NaCl) was monitored continuously
over 48 h. No significant change was observed in the
intensity of the charge transfer band at 450 nm.
amide (PaPy H, H is the dissociable amide hydrogen)
2.3. Activation of papain
3
have been shown to release NO upon illumination with
visible (in case of Fe and Mn) or UV (in case of Ru)
light. In this paper, we demonstrate that NO photore-
leased from [(PaPy )Mn(NO)](ClO ) (1) upon illumina-
tion with visible light (tungsten lamp, 50 W) binds to
papain (most possibly to Cys25) and causes inhibition.
The reaction is very clean and yields no other side prod-
ucts. The efficiency of binding to Cys25 is dependent on
both the exposure time and the concentration of the me-
tal nitrosyl inhibitor. Complex 1 thus acts as a very spe-
Twice-crystallized papain was activated by stirring
the enzyme in 50 mM phosphate buffer (pH 6.2) con-
taining 10 mM dithiothreitol (DTT) for 24 h. The solu-
tion was then passed through a Pharmacia 20 mL
desalting column equipped with a BioRad FPLC absor-
bance/electrical-conducting detector at 4 ꢁC. The acti-
vated protein (devoid of DTT) eluted from the column
in 2 min at a flow rate of 2 mL/min, was subsequently
lyophilized and stored at ꢀ20 ꢁC.
3
4

1460
R.K. Afshar et al. / Journal of Inorganic Biochemistry 99 (2005) 1458–1464
2
[
.4. Papain inhibition studies using
(PaPy )Mn(NO)](ClO )
Spectrometer. Protein samples collected from the HPLC
were injected directly into the Z-spray source of the MS
at a flow rate of 20 lL/min. The instrumental parame-
ters used were as follows: cone voltage 30 V, capillary
voltage 2.90 kV, multiplier 550 V, desolvation temp
110 ꢁC, and cone block temp 150 ꢁC. The mass spectra
were deconvoluted using MaxEnt 1 software (Waters
Micromass).
3
4
Papainactivity wasdeterminedspectrophotometrically
at 410 nm by monitoring the formation of p-nitroaniline
from the substrate N -benzoyl-L-arginine-p-nitroanilide
a
(L-BApNA). A 1.5 mM stock solution of L-BApNA was
prepared in 50 mM phosphate buffer (pH 6.2, 1 mM
EDTA, 0.1 M NaCl) and stored at room temperature. A
4
.6 lM stock solution of active papain was also prepared
in 50 mM phosphate buffer (pH 6.2, 1 mM EDTA, 0.1 M
3. Results and discussion
NaCl) and stored at 0 ꢁC. Finally, a freshly prepared
3
.0 mM stock solution of 1 was prepared in 50 mM phos-
The manganese nitrosyl complex 1 is very stable in
the buffer solution (pH 6.2) in the absence of light. How-
ever, exposure to visible light of low intensity promotes
photorelease of NO and a Mn-containing red intermedi-
ate which eventually affords Mn(III)-phosphate. This
phate buffer (pH 6.2, 1 mM EDTA, 0.1 M NaCl) and
stored in the dark.
To initiate inhibition, 250 lL of active papain was
added to 50 lL of a serially diluted solution of 1 (final
concentration ranged from 0.083 to 0.500 mM) in the
dark in a 650 lL cuvette. The mixture was then exposed
to a 50 W tungsten bulb [38] for 60 s and 350 lL of the
substrate was quickly added. The residual enzyme activ-
ity was monitored at 25 ꢁC.
To measure light dependence on the transfer rate of
NO to active papain, the exposure time was varied.
The concentrations of the substrate and enzyme were
kept the same as above and the final concentration of
6
behavior of 1, a {Mn–NO} nitrosyl, contrasts that of
6
2+
the corresponding {Fe–NO} species [(PaPy )Fe(NO)]
3
which rapidly loses NO upon dissolution in aqueous
phosphate buffer (pH 6.2) even in the absence of light
(evidenced by the loss of the charge transfer band at
6
501 nm). The inherent instability of the {Fe–NO}
nitrosyls derived from polypyridyl ligands namely
2
+
2+
[(PaPy )Fe(NO)] , [(PcPy )Fe(NO)] , and [(MePcPy )-
3
3
3
2
+
Fe(NO)] (where PcPy H is N,N-bis(2-pyridylmethyl)-
amine-2-N -(2-pyridinylmethyl)acetamide,andMePcPy H
is N,N-bis(2-pyridylmethyl)amine-2-N -[1-(2-pyridinyl)-
3
0
1
was maintained at 0.167 mM. A 250 lL aliquot of ac-
3
0
tive papain was added to 50 lL of 1 and the mixture was
exposed to a 50 W tungsten lamp for various time inter-
vals (0–90 s). After each exposure, 350 lL of the sub-
strate was added to the solution and the residual
enzyme activity was monitored at 25 ꢁC.
ethyl]acetamide) in aqueous medium has been reported
6
by us in a recent paper [38]. All these {Fe–NO} nitros-
yls rapidly lose NO in aqueous media, a reaction that we
have exploited to nitrosylate penicillamine, 3-mercapto-
propionic acid and glutathione (GSH). Although such
trans-nitrosylation reactions are very desirable, we could
not control the NO transfer process by the use of light.
2.5. Separation and identification of papain–SNO adduct
using high-pressure liquid chromatography
6
With the present {Mn–NO} nitrosyl 1, such control is
achieved. A mixture of 1 and GSH in aqueous buffer
is quite stable in the absence of light. However, upon
illumination, one obtains nitrosylated glutathione
A mixture of active papain (3 mg/mL, 1 mL) and 1
3 mM, 1 mL) in 50 mM phosphate buffer (pH 6.2,
.1 mM EDTA, 0.1 M NaCl) was exposed to a 50 W
(
0
(GSNO) in 25% yield within 1 min.
tungsten lamp for 3 min at 25 ꢁC. The enzymatic activity
was completely eliminated. The protein was then in-
jected into a Spectra-Physics UV 2000 HPLC setup with
a Grace VYDAC reverse phase C4 column (10 lm par-
ticle size, 4.6 · 150 mm, 1 mL loop volume). The elution
In recent years, various groups have studied the inhi-
bition of papain, a cysteine protease, by various NO do-
nors such as GSNO, glucose-SNAP-2 and dephostatin
[
32,39]. The rate of inhibition depends on the decompo-
sition rate of the S-nitrosothiols and, in all cases, the
protein is S-thiolated (at Cys25). Thus, the inhibition
is primarily caused by the thiyl radicals formed during
decomposition of the S-nitrothiols and not by NO
was carried out using the following gradient of H O–
2
MeCN: 95%:5% H O:MeCN to 75%:35% H O:MeCN
2
2
in 5 min, then to 45%:55% H O:MeCN in 60 min, and
2
finally to 5%:95% in 3 min. The retention time of both
active papain and papain–SNO was 40.7 min under
these conditions.
[
(
1
32]. The photo behavior of 1 prompted us to study
a) whether inhibition of papain could be achieved by
, (b) whether the extent of inhibition could be con-
trolled by light and (c) whether the presence of
papain–SNO adduct could be identified.
2
.6. Mass spectrometry
The inhibition of papain has been studied by the use
of the chromophoric substrate N-a-benzoyl-L-arginine-
p-nitroanilide (L-BApNA). The assay is based on
Electrospray ionization mass spectrometry (ESI-MS)
was carried out on a Waters Micromass ZMD Mass

R.K. Afshar et al. / Journal of Inorganic Biochemistry 99 (2005) 1458–1464
1461
p-nitroaniline (pNA), one of the hydrolytic products of
L-BapNA, that exhibits an intense yellow color
The residual activity of papain was analyzed using
the Kitz–Wilson plot [40] and the result is shown in
Fig. 2. The linear relationship between 1/kobsd and 1/
[I] indicates a bimolecular process dependent on the
photolability of 1. With the assumption of NO as an
irreversible inhibitor (the remaining metal complex does
not bind to the active site), K and k were calculated to
be 2.46 mM and 64.8 min , respectively. Although 1 is
very soluble in aprotic solvents like MeCN and DMF
and hence readily associates with the enzyme to form
ꢀ
1
ꢀ1
(
kmax = 410 nm, e410 = 8800 M cm ) in pH 6.2 phos-
phate buffer. Incubation of papain with 1 in the dark re-
sults in no inhibition as the rate of hydrolysis remains
unchanged. However, when the reaction mixture is ex-
posed to a 50 W tungsten lamp, rate of hydrolysis drops
remarkably. In one set of experiment, mixtures of pa-
pain (4.6 lM) and various concentration of 1 (in the
range of 0.083–0.500 mM) were exposed to the same
light for 60 s and the rates of hydrolysis of L-BApNA
by the irradiated protein were measured. The rate of
inhibition was then analyzed in terms of Eq. (1), where
K and k represent the dissociation constant for the en-
I
i
ꢀ1
the initial E:I complex, the K value is larger than that
I
noted for most of the thiol-containing NO donors and
the peptidyl N-nitrosoanilines [32,39]. This is quite ex-
pected since the relative affinity of the latter NO donors
with peptidyl and/or multiple aromatic groups toward
the active site of papain is higher than that of the nitro-
syl 1. Despite this fact, one must note that the overall
I
i
zyme–inactivator complex (E:I, I = 1) and the inactiva-
tion rate constant, respectively.
k1
ki
ꢀ
1
E þ I ꢀ E : I ! E ꢀ I
k
i
¼ kinactðK
I
¼ kꢀ1=k
1
Þ;
ð1Þ
ð2Þ
ð3Þ
inactivation rate k of 1 (64.8 min ) is noticeably higher
i
kꢀ1
than that of the other reported NO donors (0.339–
ꢀ
1
0
.087 min ). Also, the second-order rate constant ki/
d½E ꢀ Iꢁ
dt
k
i
½Iꢁð½Eꢁ ꢀ ½E ꢀ IꢁÞ
0
v ¼
¼
;
6
K for the inactivation of papain by the {Mn–NO}
I
K
I
þ ½Iꢁ
4
ꢀ1 ꢀ1
nitrosyl 1 is 2.63 · 10 M
tially greater than the k /K
and N-nitrosoanilines reported by Wang and coworkers
32,39]. The superior inactivation capacity of 1 clearly
s
. This value is substan-
values for the S-nitrosothiols
I
1
K
I
1
i
¼ k þ k
.
k
obsd
i
½Iꢁ
i
[
The overall rate expression for the formation of S-
arises from the rapid photogeneration of NO from 1
nitrosylated protein (E ꢀ I) is given in Eq. (2), and the
relationship between the observed pseudo first-order
rate constant (kobsd) and the concentration of the inacti-
vator is shown in Eq. (3). The extent of inhibition as a
function of the inhibitor concentration is shown in
Fig. 1.
compared to the slow RS–NO bond homolysis at
25 ꢁC in case of the latter NO donors. Since NO does
not recombine with the Mn(III) photoproduct (vide in-
fra), the inhibition of papain by 1 is very effective. We
are aware of the fact that part of the discrepancy could
be related to the use of L-BApNA versus N-benzyloxy-
carbonyl-glycine-p-nitrophenyl ester as the substrate
[
41]. Nevertheless, the inhibition of papain by 1 is
0
0
0
0
0
.8
.6
.4
.2
.0
remarkable since the inhibition is observed only when
the reaction mixture is exposed to light. It is important
to note that the photogenerated NO is able to cause
a
b
c
d
7
6
5
4
3
2
1
0
e
f
0
200
400
600
800
Time (s)
Fig. 1. Light-dependent inactivation of papain by the photolabile
nitrosyl [(PaPy )Mn(NO)](ClO ) (1). Papain (0.3 mg/mL, 250 lL) was
treated with 1 (0.083–0.500 mM, 50 lL) at 25 ꢁC in 50 mM phosphate
3
4
0
2
4
6
8
10
12
14
buffer, pH 6.2, 1 mM EDTA, and 0.1 M NaCl while illuminated with a
-
3
-1
5
0 W light bulb for 60 s. The substrate, L-BApNA (0.560 mM, 350 lL),
was then added and the formation of pNA was measured at 410 nm at
s intervals. (a) 0.083 mM of 1; (b) 0.167 mM of 1; (c) 0.250 mM of 1;
d) 0.333 mM of 1; (e) 0.417 mM of 1; (f) 0.500 mM of 1.
1
/[I] x 10 (M )
1
(
Fig. 2. Kitz–Wilson plot for the reaction of papain with 1 in 50 mM
phosphate buffer (pH 6.2, 1 mM EDTA, 0.1 M NaCl) at 25 ꢁC.

1462
R.K. Afshar et al. / Journal of Inorganic Biochemistry 99 (2005) 1458–1464
S-nitrosylation before oxidation to NO under aerobic
not arise from any unidentified interaction between the
metal complex and the enzyme. Again, the rate of
2
conditions. The generation of an S-nitroso species at
the active site of papain requires an electron to be re-
moved. We believe that dioxygen accepts this electron.
Dioxygen is also responsible for the generation of the
Mn(III) photoproduct [37].
A time-dependent study was also performed to deter-
mine the effect of the exposure time on the process of
inhibition. As shown in Fig. 3, the release of NO from
a 0.167 mM solution of 1 and the consequent inhibition
of papain were directly dependent on the exposure time.
As the exposure time was increased, the rate of hydroly-
sis of the substrate (present in excess) by the protein de-
creased almost linearly.
hydrolysis was not affected by the presence of [(Pa-
+
Py )Mn(Cl)] in the reaction mixture. It is thus clear
3
that inhibition of papain by 1 under light occurs due to
S-nitrosylation reaction only; no secondary interaction
between the enzyme and the metal-containing remains
is responsible for the observed inhibition. This is in con-
trast to the formation of the S-thiolated products
formed during inhibition of papain by the S-nitrosothi-
ols [32].
In the present work, the formation of the S–NO ad-
duct in papain has been confirmed by HPLC–MS. In
this experiment, a mixture of papain and 1 was first ex-
posed to light for 2 min and then stirred for an addi-
tional 20 min. The solution was then loaded onto a C4
reverse phase HPLC column and the fraction with reten-
tion time of 40.7 min was collected. This purification
step allowed removal of both the Mn–phosphate end
We have previously shown that the light-induced
photolysis of 1 in MeCN under aerobic conditions af-
2
+
fords the solvated species [(PaPy )Mn(MeCN)] [37].
3
In presence of H O, the labile MeCN-adduct forms
2
2
+
the reddish brown l-oxo species [{(PaPy )Mn} O]
via a [(PaPy )Mn(H O)] intermediate, which has a
3
2
2
+
3
2
short lifetime in aqueous solution. In aqueous phos-
phate buffer (pH 6.2, 1 mM EDTA, 0.1 M NaCl), the
photolysis of 1 results in a red solution whose composi-
tion is unknown at this time. To ensure that the end
product(s) of the photolysis reaction did not interact
or chemically modify papain through some secondary
reaction, we have examined the rates of hydrolysis of
the same substrate by papain in the presence of the
photolyzed product(s). No change in the rate of L-BAp-
NA hydrolysis by papain was observed upon addition of
a photolyzed solution of 1. The rate of L-BApNA hydro-
23429
1
00
lysis was also examined in the presence of [(Pa-
+
Py )Mn(Cl)] [37] to ensure that papain inhibition did
3
0
23459
1
00
0
0
0
0
0
.8
.6
.4
.2
.0
1
0 s
2
0 s
3
0 s
6
0 s
9
0 s
0
0
200
400
600
800
20000
22000
24000
26000
28000
30000
Time (s)
Mass (m/z)
+
Fig. 3. The light-dependent inactivation of papain by the photolabile
nitrosyl [(PaPy )Mn(NO)](ClO ) (1). Papain (0.3 mg/mL, 250 lL) was
Fig. 4. Top panel: mass spectrum of Na [papain–SH] protein. Bottom
+
3
4
panel: mass spectrum of Na [papain–SNO] protein showing the
treated with 1 (0.167 mM, 50 lL) at 25 ꢁC in 50 mM phosphate buffer,
pH 6.2, 1 mM EDTA, and 0.1 M NaCl while illuminated with a 50 W
light bulb at various time intervals (10–60 s). The substrate, L-BApNA
addition of 30 mass units indicative of S–NO bond formation. Samples
were directly infused into the Z-spray source of the mass spectrometer
at a flow rate of 20 lL/min. Instrumental parameters: capillary voltage
2.9 kV, cone voltage 30 V, multiplier voltage 550 V, desolvation temp
110 ꢁC, and cone block temp 150 ꢁC.
(0.560 mM, 350 lL), was then added and the formation of pNA was
measured at 410 nm in 1 s intervals.

R.K. Afshar et al. / Journal of Inorganic Biochemistry 99 (2005) 1458–1464
1463
Acknowledgments
Papain
SH
+
NO
Experimental assistance from Dr. Kaushik Ghosh,
Aura A. Eroy-Reveles and Pilgrim J. Jackson is grate-
fully acknowledged.
N
N
N
Mn
N
Papain
SNO
N
hν
O
+
References
3
Scheme 1. S-nitrosylation of papain by [(PaPy )Mn(NO)] .
[
[
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product as well as the excess salts. The electrospray mass
spectra of native papain (purified by the same HPLC
method) and the nitrosylated papain fraction were ob-
tained in the positive ion mode and the m/z values of
the parent ions were calculated by the MaxEnt program
by Waters. As shown in the top panel of Fig. 4, papain
exhibits its molecular ion peak at m/z = 23,429. Crystal-
lographic results have assigned a molecular weight of
[
[
[
[
2
3,406 to pure papain [41]. The difference of 23 m/z cor-
[
[
+
responds to a Na ion that associates with papain in our
mass spectrum. The bottom panel of Fig. 4 displays a
molecular ion peak at m/z = 23,459 and corresponds
to the papain–SNO adduct (a difference of 30 mass
units). The peak at m/z = 23,459 was also obtained
when papain was incubated with GSNO (for 30 min)
and the reaction product was analyzed by mass spec-
trometry following purification by HPLC. In the latter
case, the mass spectrum showed peaks for both the pa-
painS–NO (fraction with retention time 40.7 min) and
papainS–SG (fraction with retention time 37.2 min) at
m/z = 23,459 and 23,735, respectively. Identification of
the papain–SNO adduct clearly demonstrates that inhi-
bition of papain by 1 under light occurs due to the nit-
rosylation of the active site cysteine (Scheme 1).
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. Conclusions
Inactivation of papain via S-nitrosylation has been
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3
4
6
NO} nitrosyl. The inhibition of the enzyme is achieved
only under visible light (tungsten lamp, 50 W) since the
nitrosyl is photosensitive and releases NO under illumi-
nation. The photoreleased NO inactivates papain most
possibly by nitrosylating Cys25 of papain. Unlike other
thiol-containing NO donors that afford mixed disulfides
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The extent of inhibition of papain is directly propor-
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