In vitro Nitrosilation of Insulin Chains A and B

Celina Santosa , Ricardo Afonsoc, Maria Pedro Guarinoc, Rita Patarrãoc, Ana Fernandesc,

João Paulo Noronhaa, Maria Paula Macedoc and Jorge Caldeiraa,b,*

a REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica, Portugal,

b Instituto Superior de Ciências da Saúde Egas Moniz, 2829-516 Caparica, Portugal

c Departamento de Fisiologia FCM-UNL Campo dos Mátires da Pátria, n. 130, 1169-056 Lisboa, Portugal

Elsevier use only: Received date here; revised date here; accepted date here

Abstract

The physiological roles of insulin and nitric oxide have been linked recently by a several studies. A diversity of insulin

chemical modifications are reported either in vivo or vitro. The S-nitrosation, the covalent linkage of nitric oxide to cysteine

free thiol is recognized has an important post-translational regulation in many proteins. Here we report the in vitro synthesis of

an S-nitrosothiol of bovine insulin A and B-chains characterized by their HPLC chromatographic behavior monitored by UV

visible spectroscopy and electron spray ionization mass spectrometry. The experimental results indicate the formation of

bovine insulin A and B-chain S-nitrosoinsulin adduct. Stability and solubility of these synthesized derivatives is described for

physilolgcal studies. The potential relation of these molecules with the previously described “hepatic insulin sensitizing

substance” (HISS) is adressed.

© 2005 Elsevier Science. All rights reserved.

Keywords: Insulin, Nitrosothiol, HISS.

1. Introduction

The impairment of nitric oxide and insulin is currently under the intense investigation [1,2,3,4]. Insulin is

a well known hormone that regulates the glucose uptake, and nitric oxide multiple physiological roles are

well documented. Nitric oxide and insulin relationship were studied in plasma [5] or endothelial [6]

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adipocytes [7] liver [8] pancreatic cells [9]. The different forms nitric oxide synthase (NOS) are believed

to mediate these process [10].

Insulin molecules have different aggregation states ranging from monomer, dimeric, hexametric or

oligomers/fibrils, depending on pH, concentration and presence of certain organic compounds or metal

ions [11,12]. Three dimensional structure and their T and R conformational states have been described in

detailed by NMR [13,14,15] and X-ray crystallography [16,17,18]. Each monomer has two polypeptide

chains A and B, with 21 and 30 residues respectively. The two chains have three disulfide bridges being

one intra A chain (A6-A11) and two others interchain (A7-B7 and A20-B19). The monomer was

proposed to be the functional unit that binds to the insulin receptor [19]. Insulin with amino acid

substitutions or deletions and chain A or B fragments obtained either by synthesis or by insulin degrading

enzyme (IDE) action have been described in terms of biological activity [20,21,22,23]. A large number

of chemical derivatives have also been reported that retarded or increased the activity after administration

time[24]. The NMR solution structures have been determined for isolated A-chain and B-chain in the

oxidized (SO3H form) or for B-chain with C9S and C19S mutated sequence, revealing that secondary

structure is retained from the native monomeric structure [25,26,27]. Reports in the literature describe

insulin molecules that have been submitted to glycation [28,29,30], phosphorylation [31], sulfitolysis

[32], and peroxinitrite [33] or hypochloriote [34] reaction.

The chemistry of nitric oxide (NO.) in water presents a rich variety of species namely HNO/NO-

/NO./NO+. Considerable analytical and computational effort have been made to characterize their

reactivity, redox potentials, pKa values, and spin states [35,36,37]. When nitric oxide is in the presence

of other molecules of physiological relevance like molecular oxygen or carbonate a even wider range of

species exists in solution. The experimental conditions of this work were restricted to anaerobic

conditions in non carbonated buffers [ 38,39,40,41,42,43].

Although insulin has no free cysteine in the native state, its disulfide cleavage in the presence of

endogenous thiols have been investigated [44]. Protein disulfide isomerase (PDI) [45] is an enzyme that

catalyse the isomerization of disulfide bridges in order to induce correct protein folding. The PDI enzyme

is active when insulin is the substrate either in vivo or in vitro [46,47]. The enzimatic activity requires the

presence of a low molecular weigth thiol like GSH that exists in the plasma at milimolar concentrations

[48]. Intrestingly PDI was recently characterized in a “NO charged state” where the presence of the N2O3

molecule (formed by NO and O2) in the interior hydrophobic domains of the enzyme is proposed [49].

Denitrosation activity of PDI of plasmatic GSNO from the points to another physiological link between

thiols and nitric oxide.

Several free thiol containing proteins and peptides have been modified by nitrosilating reagents

producing nitrosothiolproteins [50,51] and some have been characterized by electron spray ionization

mass spectrometry [52,53]. This post-translacional modification is widely reported in vitro and in vivo

conditions, and is responsible for a wide variety of physiological roles. Although controversly exist on

the physiological amounts of nitrosothiols due to dificulties of analytical determinations of these

compounds (namely GSNO) in biological samples [54, 55].

This study is potentially relevant for the study of the physiological impairment of insulin action and nitric

oxide in the context of Type II, non insulin-dependent Diabetes Mellitus (NIDDM). Previously some of

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us proposed the existence of a so called “hepatic insulin sensitizing substance” (HISS) where nitric oxide

donors can reestablish insulin activity during rapid insulin sensitivity test (RIST) [56] after

pharmacological of chirurgical induction of insulin resistance [57,58,59,60,61,62,63,64,65,66,67,68,69].

2. Materials and Methods.

For the in vitro nitrosilation of the protein, bovine insulin (Sigma) was used without further purification.

All other reagents were analytical grade or higher. Nitrosilation was performed after disulfide reduction

by two alternative methods of nitrosilation.

Reduction of Insulin disulphide bridges

Reduction of Bovine Insulin (10 mg/ml in a HEPES 25mM solution, pH 8.2) was performed with

Mercaptoethanol 6.9 mM in the presence of 8.0 M urea and 1.0 mM EDTA in anaerobic conditions for

one hour at room temperature in the dark. No thiol cysteine alquilating agents were added. Nitrolisation

were performed immediately after the reduction process.

Reaction of Insulin with H+/NO2-

Reduced insulin samples of 100 µl with mercaptoethanol, EDTA and urea were incubated with HCl or

HCOOH (final concentration 0.3-0.7M) and NaNO2 (final concentration 0.045-0.297M) for 10 minutes.

The reaction terminated by neutralizing the solution by the addition of NaOH (final concentration 0.3-

0.7M) and immediately injected into the chromatographic column for peptide separation and analysis.

Reaction of Insulin with authentic NO gas

Reduced insulin samples of 100 µl with mercaptoethanol, EDTA and urea were reacted with a gentle

stream of nitric oxide/argon gas, 5:95 mixture (Arliquide) for 30 minutes in the dark at 4ºC. NO/Ar gas

was bubbled first in to a 10M KOH solution and then to millipore water in order to remove NOx traces.

Electrophoresis

Samples of native insulin, and chromatographically purified A-chain and B-chain were applied in to a

20% SDS-PAGE gel. Samples buffer used didn’t contain mercaptoetanol in order to evaluate the eventual

multimerization upon oxidation, due to intermolecular difulfide bridge formation, of the purified A and

B-chain in the SH form.

Cromatography of the reaction mixtures.

Chromatographic analysis was carried out using a Merck L-7100 HPLC equipped with an L-7400 UV

detector and D-7000 computer interface. Alternatively for PDA detection a ChromQuest

Chromatography Data System (Finnigan Surveyor plus HPLC) equipped with LightPipe™ PDA was

used for online recording of the UV spectra.

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Reversed phase C18e column (250 x 4 mm Sperisorb from Merck KGaA - Darmstadt, Germany) was

used with the mobile phases A (water with 0.05% TFA) and B (acetonitrile with 0.05%TFA). The elution

gradient was as follows (flow rate 1.0 mL/min): 0-5 min, 5% B; 5 – 40 min, 95% B.

Alternatively size exclusion chromatography (Superdex HR 10/30 Peptide Pharmacia) of reaction

mixtures were performed with detection at 280 nm or in DAD detection from 200 to 600 nm with a flow

0.9 mL/min using a well degassed mobile phase with typically 2% formic acid (for positive ionization

mass spectrometry analysis) or 1% ammonia solution (for negative ionization mass spectrometry

analysis). Chromatographic peaks were collected manually and partially dried in a speed vac (Braun). For

HPLC-ESI-MS analysis, mobile phases were acetonitrile + formic acid 1% (50:50 by volume) and

acetonitrile : ammonia 1% (50:50 by volume) for positive and negative ionization mode, respectively, at a

flow rate of 0.9 mL/min.

Electron Spray Ionization Mass spectrometry

Masses of peptides were determined using ESI. Partially dried samples were dissolved in 50:50

water/acetonitrile containing 1 % formic acid, and directly injected to the electron spray mass

spectrometer (MicroLynx 4.0 SP1) with a 50 µL/min flow. For HPLC-ESI-MS analysis, an accurate

splitter (split ratio of 1:100) was used between the HPLC column (flow rate 0.9 mL/min) and the mass

spectrometer.

Capillary temperature were kept at 100ºC or 120ºC using a cone voltage of 35 V or 50V and capillary

voltage of 3.5 KV , and spectra mass/charge range 500-2000 Da.

.

3. Results and Discussions

Reduction disulfide bridges of bovine insulin are easily observed by increase in turbidity of insulin

solution (attributed to chain B precipitation) in the absence of a denaturant agent such as urea. When the

reaction is performed in the presence of 8M urea, a clear solution is observed. Upon nitrosilation either

with H+/NO2- or NO: Argon (5:95%) gas a pink color solution is observed characteristic of nitrosothiol

compounds with a UV/Visible band at ~350 nm (ε = 890 M-1cm-1) and a weaker band at 545 nm (ε = 16

M-1cm-1)refannenglish, due to the formation of both peptide (A and B-chain) and mercaptoethanol

nitrosothiols (HO(CH2)2SNO) B. However, when a small concentration of mercaptoethanol was used (6.9

mM) a yellowish solution was produced with an absorption maximum at 330-350nm. Nitrosilation

immediately after disulfide reduction, without separation of the reaction mixture, prevents the

aggregation, as observed by size exclusion chromatography, and SDS-Page electrophoresis where the

two major peaks with the corresponding retention time as A and B-chain either in SH or SO3H form

were observed (see Figure 1).

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The two peaks that appeared on chromatogratogram to the presence of bovine insulin A-chain (retention

time 15.4 min) and B-chain (retention time 13.4 min). Under the same conditions monomeric insulin (low

pH) of 11.9 min and other molecules from the reaction mixture (EDTA, urea and Hepes) at retention

times higher that 20 min (not showed). The difficulties in the preparation of the nitrosilated chain A and

B are the strong propensity to aggregate based on strong hydrophobic interchain association together with

the tendency to form interchain disulfide bridges, yielding insoluble high molecular weight aggregates.

Aged preparation (overnigth) yield high molecular aggregates as judged by SDS-PAGE electrophoresis

or by size exclusion chromatography (retention time 8.1 min) in denaturating conditions (70% formic

acid) (not showed).

3.1 Chromatography

Several HPLC methods are established in the literature [70,71] including the European pharmacopoeia

protocol [72] to quantify insulin and their degradation products [73]. However these rely on mobile

phases with high salt concentrations not suitable for mass spectrometry and preparative purposes.

Reaction mixture was initially submitted to RP C18e (and C8) HPLC chromatography using a linear

gradient of water/acetonitrile with 0.05% TFA. This purification step allowed to identify peaks

corresponding to nitrosilated mercaptoethanol and A-Chain insulin (SH and SNO form) and B-Chain

insulin (SH and SNO form), Fig. 2. However reversed phase column (C18e and C8) had a low recovery

of the purified peptides. For this reason, size exclusion chromatography with denaturant eluent [74,75]

was adopted as a preparative method. Although SH and SNO forms of B-Chain and A-Chain could not

be separated due to small molecular mass difference, they could both be clearly identified in the ESI-MS

spectra.

Size exclusion chromatography had the advantage (besides good recovery) that can be performed in the

denaturating (formic acid from 70 to 2 %) and that 50 - 100 µM nitrososothiol mercaptoethanol could be

added to the eluent to prevent decomposition of the nitrosothiol peptides during preparative

chromatograpy [76,77]. Inset in Figure 1 shows the online UV-visivle spectra for insulin A-chain (peak

2) is shown (similar for insulin B chain). Nitrosilated insulin A-chain was demonstrated since an increase

in the absorbance band at 320nm was observed.

3.2 Electron Spray Ionization Mass Spectrometry

ESI-MS was used to probe nitrosilation products of reduced insulin. Insulin B-chain gave protonated

precursor molecular ions [M+H]+ in the positive ESI MS mode. On the other hand, insulin A-chain was

better observed in negative ionization mode. For this reason, a trace of formic acid was added to the

sample or to the mobile phase (when HPLC-MS analysis were performed) to aid protonation for positive

ionization mode and a trace of ammonia solution for negative ionization mode. For direct samples

injection on ESI-MS equipment samples were only partially dried because vacuum shifts the equilibrium

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RSNO ⇔ RSH + NO(gas). Peaks spectra (after deconvolution) of insulin B-chain in the SH and SNO

forms is presented in Figures 2 and 3 with the approximate component ratio of 5:1. Peak at M = 3399 Da

corresponds to the reduced bovine insulin B chain (M = 3399.9 Da). Peak at M = 3428 Da corresponds to

the expected mass of reduced insulin B chain after addition of a NO group (+ 29 Da) and thiol

deprotonation. The bovine insulin B-chain NO adduct was found to be sensitive to both speed vac

complete dryness or to high capillary temperatures which are compatible observations expected to the

labile nature o nitrosothiols.

After separation by HPLC the mass spectra for insulin A chain and A-chain NO aduct can be observed

in negative ionization mode (see Figure 3). Decovolution of the negative ESI-MS spectra reveal peaks at

M = 2339 Da corresponding to the reduced (SH) insulin A-chain (M = 2339.6 Da) and peak at M = 2368

Da corresponds to the expected mass of bovine insulin A-chain after addition of a NO group and thiol

proton loss. In both A or B-chain only the mono nitrosilated products were observed.

Physiological implications

The relation between insulin action and nitric oxide has been intensively studied recently. Some of us

have proposed the existence of a so-called HISS hepatic insulin sensitizing substance. This hypothetical

substance was proposed based in vivo studies were rats with induced type II diabetsis could regain

insulin sensitivity by addition of NO donor during the RIST test. So far there is no direct evidence that

insulin or insulin chains exists has a NO adduct in vivo. The detail knowledge of the stability and

behavior during purification protocols of these forms in vitro is a mandatory requirement to detect

possible labile nitrosthiol peptides in vivo. It is well known that smaller peptides of the insulin chain A

and B have significant biological activity. Also structural studies of insulin B chain in the oxidized

(SO3H form) retain most of the structure observed in the monomeric/hexameric form determined by

NMR and X-ray crystallography. With this study we addressed the requirements in terms of stability due

to formation of oxidized and or multimeric forms. This stability is directly linked to the resulting

solubility of the polypeptide. Also we observed that insulin chain B can be soluble with well degassed

pysiological soro with 1mM GSH compatibe with intravenous administratuion. This open the posibility

to explore a new types of insulin derivatives for immunoassays (RIA) and in vivo testing.

Acknowledgments

This work was supported by POCI/SAU-OBS/56716/2004 (M.P.M.) and POCTI/QUI/58973/2004

(J.C.) FCT-MCTES grants.

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Figures

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 2 4 6 8 10 12 14 16 18

Retention Time (min)

Inte

nsity

(AU

)

Figure. 1. Size exclusion chromatography separation of reaction products formed after nitrosilation of

reduced bovine insulin with H+/NaNO2. Peaks: 1 = insulin B-chain and its NO aduct ; 2 = insulin A-chain

and its NO aduct. Inset shows the online UV-visible spectra of peak 2 with characteristic nitrosothiol

aduct absorbance.

0

0,5

1

250 300 350 400 450 Wavelength (nm)

Abs

orba

nce

2

1

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0

50

100

3360 3370 3380 3390 3400 3410 3420 3430 3440

Mass (Da)

Rel

ativ

e Ab

unda

nce

Figure 2- Deconvolved electrospray mass spectra of the insulin after reduction with mercaptoethanol

and nitrosilation with H+/NaNO2.

0

50

100

2310 2320 2330 2340 2350 2360 2370 2380 2390

Mass (Da)

Rel

ativ

e Ab

unda

nce

Figure 3- Deconvolved electron spray ionization mass spectra of the insulin after reduction with

mercaptoethanol and nitrosilated with H+/NaNO2 .

3399 Insulin B chain SH

3428 Insulin B-chain SNO

2339 Insulin A-chain SH

2368 Insulin A-chain SNO

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