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A NEXAFS and mass spectrometry study of cysteine, cystine and insulin irradiated with intermediate energy (0.8 keV) electrons

https://doi.org/10.1016/j.elspec.2014.02.002Get rights and content

Highlights

  • Structural modifications in sulfur containing biomolecules were investigated.

  • Significant modifications were observed in insulin irradiated NEXAFS spectra.

  • Degradation of insulin can be observed even at low temperature.

  • Alterations in insulin spectrum were characterized according to the state of sulfur.

Abstract

We have performed a NEXAFS (S 1s) and mass spectrometry study of solid samples of cysteine, cystine and insulin irradiated with 0.8 keV electrons. The measured mass spectra point out to processes of desulfurization, deamination, decarbonylation and decarboxylation in the irradiated biomolecules. Electron beam irradiation was also conducted at low temperatures in order to evaluate the possible contribution from thermal degradation processes. The NEXAFS spectra of irradiated cysteine and cystine did not show substantial changes when compared to the same spectra obtained using non-irradiated samples. The sulfur K-edge photoabsorption spectrum of irradiated insulin, however, showed clear modifications when compared to the spectrum of the non-irradiated protein, even when the irradiation was conducted at low temperature. Using an empirical combination of the photoabsorption spectra of cysteine and cystine (which are associated respectively with reduced and oxidized forms of sulfur) we have been able to reproduce the absorption spectrum of irradiated insulin.

Introduction

The physical and chemical damage induced by electrons may severely limit the amount and quality of information which can be obtained about biomolecules using important experimental techniques such as electron microscopy, electron diffraction, X-ray microscopy and X-ray diffraction [1], [2]. When electrons are the primary interacting particles (electron diffraction, electron microscopy, electron energy-loss spectroscopy and so on), inelastic scattering will give rise to other fast electrons as well as to radicals, ionic species and low energy (<30 eV) secondary electrons. The latter are considered to play a decisive role in the chemical damage of biological samples and as a result have been the focus of many important studies [3 and references therein]. With photons in the 0.1–12 keV energy range, photoemission becomes the dominant reaction channel and again fast and slow electrons will be produced and interact with the samples. We recall that 12 keV corresponds roughly to a photon with 1 Å wavelength, widely used in synchrotron radiation centers, in X-ray chrystallography protein studies [4]. At the same time, considering the main atomic constituents of amino acids, proteins and nuclei acids (carbon, nitrogen, oxygen and sulfur) we realize that core-level photoabsorption (NEXAFS) studies will in general give rise to slow (less than 100 eV) and intermediate energy electrons (kinetic energies in the hundreds of eV to a few keV range). It should be emphasized that while low energy electrons basically interact with the surface of the samples, intermediate energy electrons show a significant damage depth. The interrelation between the terms penetration depth, attenuation length and damage depth caused by intermediate (100–1000 eV) electrons is still a subject of much interest and much discussion. For instance, Lamont and Wilkens have shown, on their study of self-assembled monolayers of alkanethiols on gold [5], that the attenuation length of 1 keV electrons should be of the order of 23 nm. In a more recent and very interesting publication, Barnett and collaborators have shown, using an electron gun, that, in the 0.1–2.0 keV energy range, damage depths in polyaromatic compounds (PAH's) increase linearly with approximately 110 nm per 1 keV [6]. It is consequently of importance to study the irradiation of biomolecules induced by intermediate energy electrons. In the present paper we use mass spectrometry and sulfur K-edge photoabsorption techniques to investigate the processes induced in three sulfur-containing biomolecules: cysteine, cystine and insulin, following irradiation with 0.8 keV electrons.

Sulfur is an important element present in biological systems and can be found in several oxidation states. In cysteine, sulfur is present in a reduced form as a thiol (single bondSsingle bondH). In cystine and insulin, sulfur is present in an oxidized form: a disulfide bond (single bondSsingle bondSsingle bond) (Fig. 1, Fig. 2). The disulfide bonds are formed by a covalent bonding between the sulfur atoms of two cysteine residues. This kind of chemical bond plays a most significant role in the definition of the molecular structure of important peptides and proteins [7].

The cleavage of disulfide bonds is one of the consequences of radiation damage to sulfur containing proteins [8]. Weik et al. investigated specific chemical and structural damage in enzymes caused by synchrotron radiation. They observed the cleavage of disulfide bridges even at cryogenic conditions [4]. Schulze-Briese et al. studied the beam-size effects of radiation damage in insulin crystals and again significant changes in disulfide bridges were observed [9].

NEXAFS and mass spectrometry have been previously used by Zubavichus et al. in the study of the decomposition of five amino acids exposed to an X-ray source. K-edge spectroscopy of carbon, nitrogen, oxygen and sulfur L2,3 edge and mass spectrometry were complemented by XPS measurements [10].

The advantages of sulfur K-edge spectroscopy have been widely discussed and are mostly due to its sensitivity as a probe to identify and quantify the chemical forms of sulfur. A NEXAFS spectrum can be used as a fingerprint to speciate sulfur metabolites even in complex biological samples. The near-edge region is dominated by dipole-allowed (Δl = ±1) bound-state transitions of the 1s electron and presents relatively sharp lines and large chemical shifts according to the sulfur oxidation states.

In the present work, mass and NEXAFS spectra have been obtained at ambient and low (77 K) temperatures for the three mentioned samples. In particular and within our knowledge, this is the first time in which NEXAFS and mass spectrometry are conjointly used to shed light on the degradation of intermediate energy electron-irradiated insulin.

Section snippets

Materials and methods

The samples – insulin (bovine), l-cysteine and l-cystine non-crystalline powders (Sigma–Aldrich, purity >98%) – were deposited without any previous treatment on a carbon sticky paper (STR tape from Shinto Paint Co.) forming a uniform layer. The tapes were fixed to an aluminum holder and attached to a high vacuum precision positioning system.

Samples were irradiated with a Kimball Physics electron gun system (EGA-1108/EGPS 1108) using an 800 eV electron beam. The electron beam diameter at the

Mass spectrometry

As it is well known, following electron irradiation of amino acids deamination and decarboxylation takes place. For sulfur-containing amino acids, desulfurization also takes place [2]. We have accordingly decided to monitor the time behavior of the partial pressures associated with the following m/z values: 16, 28, 34, 44 and 64. The m/z value of 16 was chosen in order to study deamination processes. It corresponds to the NH2+ ions although it may contain a contribution from O+. The m/z = 17 (NH3+

Conclusion

Degradation processes in solid samples of cysteine, cysteine and insulin, subjected to a 0.8 keV electron beam have been studied using the NEXAFS and mass spectrometry techniques. Measurements were made both at room and at low temperature (77 K). The emission of gases associated with degradation processes such as deamination, decarboxylation and desulfurization of the samples was observed even at very low temperatures, following electron beam irradiation.

Changes in the sulfur oxidation state,

Acknowledgments

The authors would like to acknowledge CNPq, FAPERJ, CAPES and LNLS for the financial support and the SXS beam line staff for technical support.

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