Characterisation of the rubber elongation factor from ammoniated latex by electrophoresis and mass spectrometry

Abstract

Rubber elongation factor (REF) is considered as one of the major allergens present in latex. An extraction and purification protocol for preparation of REF standards has been modified. A protein fraction was extracted from ammoniated latex sap and purified by gel filtration chromatography. The purified and concentrated proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis into two major bands. These bands were further characterised by matrix-assisted laser desorption/ionisation time-of-flight and nano–electrospray ionization mass spectrometry. REF and a truncated form could be ascertained by the mass and fragmentation pattern of the tryptic peptides. In the faster migrating band an additional peptide could be identified. This peptide is also present in Hevb3 and a M_r 27 000 latex allergen. Our findings indicate that conventional REF preparations as standards may contain additional allergenic proteins.

Introduction

In the past two decades the immediate type of latex allergy has become a serious problem for an increasing number of individuals. At high risk for this type I allergy against latex are patients with spina bifida, patients who have undergone a number of surgeries in the genitourinary tract and individuals who are frequently exposed to latex products [1], [2], [3], [4], [5], [6], [7]. Three to ten percent of health care workers are allergic against components of different latex products [6], [8], [9]. Yassin et al. [10] have predicted an even higher prevalence of IgE-mediated latex allergy of approximately 17%. The symptoms range from a minor skin rash to anaphylactic reactions causing the death of a patient [11], [12]. According to the significance of this problem, a number of allergens present in latex were identified in the last few years. The main allergens in latex products such as gloves are no longer the additives required for production. Proteins originating from the rubber tree Hevea brasiliensis are considered as potential allergens. Two proteins are regarded as the most crucial allergens in latex products; namely the rubber elongation factor Hev b1 (REF) [13], [14], [15], [16], [17], [18], [19] and Hevein [20], [21], [22], [23], [24], [25]. REF is a highly hydrophobic M_r 14 600 protein located on the surface of the rubber particles. Hevein is the M_r 4700 subunit of Prohevein, a wound-induced protein in the laticifers of the rubber tree.

Purified REF is frequently used in various test systems for diagnosis of latex allergy. Common to many protocols for preparation of an extract representing all soluble proteins from ammoniated latex sap is a batch extraction with phosphate-buffered saline (PBS) or Tris buffer. REF, as a highly hydrophobic protein, is usually isolated from this protein extract by an additional extraction with an sodium dodecylsulfate (SDS)-buffer followed by gel filtration [15], [18] or SDS–polyacrylamide gel electrophoresis (PAGE) combined with electroelution [13].

Our interest lies in the area of identification of allergenic proteins present in latex gloves. Latex itself is a common and rich source to extract this proteins as reference for structural and biological studies. For our investigations, the extraction of REF was performed as previously described by Czuppon et al. [15] while the purification step was modified. The dialysis step was omitted and an additional gel filtration with a Sephadex G 25 column combined with a discontinuous ultradiafiltration to concentrate the diluted eluates was added. Further purification was achieved by gel filtration with a Superdex 75 column as described by Czuppon et al. [15]. The eluates was lyophilized.

The conventional separation on SDS–PAGE in combination with the mass spectrometric methods of nano–electrospray ionization (nano–ESI) or matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry introduces a new dimension for identification of these proteinogenic allergens. The separated proteins can be directly eluted from the gel [26] or can be measured after in-gel digestion with endoproteases [26], [27]. These methods offer the possibility of peptide mapping as well as the characterization and identification of these molecules by aligning the sequence of the peptides to the sequences of known proteins.

Proteins present in ammoniated latex sap have been extracted and purified by our modified procedure. The proteins have been partially characterized either directly from bands in SDS–electrophoresis gels by MALDI–TOF-MS or after tryptic in-gel digestion by nano–ESI-MS.