No abstract

The interaction of benzyl isothiocyanate (in concentrations of 2·5–250 mg benzyl‐ITC g−1 protein) with myoglobin leads to the formation of derivatives which have been characterised in terms of their solubility, free epsilon amino groups, content of tryptophan and its quenching, as well as their molecular properties determined with electrophoretic and chromatographic methods. The reaction takes place primarily at the epsilon amino groups of lysine, whose content decreases depending on the concentration of the benzyl‐ITC present. A second possibility is the reaction of the secondary amino group present in tryptophan, whose concentration also decreases. These reactions increased the electrophoretical mobility during PAGE in the presence of urea, as a result of which the isoelectric points shifted from 7·33–7·45 to 5·13–5·6 as shown by means of the isoelectrical focusing technique. The molecule size (size exclusion chromatography) decreased as a result of increased hydrophobicity (RP‐HPLC). A slight polymerisation with an increase in the content of dimer (38 kDa, SDS‐PAGE) was observed. The derivative with the highest degree of derivatisation (250 mg benzyl‐ITC g−1 protein) showed an unexpected behaviour with all its investigated properties, which can be explained as a result of structural changes taking place during the process of derivatisation.

SummaryGlucosinolates and their very highly reactive breakdown products (mainly isothiocyanates = ITC) belong to one of the most important natural toxicants. This paper deals with the interactions of allyl-, butyl-, phenyl-and benzyl ITC with egg white proteins resulting in the formation of ITC-proteinconjugates. The ITC's react with amino groups by forming thiourea derivatives. Such reactions are well described by changes in the amounts of free amino groups and available lysine. Further reaction sites are sulphhydryl side chains of proteins leading to dithiocarbamate esters. The investigations further show that such reactions are accompanied with a decrease in the solubility of the prepared conjugates. Simultaneously a shift of the isoelectric range to a lower value of pH can also be observed. Changes in electrophoretic patterns and mobility can also be seen due to the strong electrophilic attack of ITC to proteins. Comparison of the 4 investigated preparations shows that benzyl-ITC is the most reactive partner for egg white proteins. ZusammenfassungGlucosinolate und ihre reaktiven Spaltprodukte (hauptsachlich Isothiocyanate = ITC) zahlen zu den wichtigsten natiirlichen Schadstoffen. Wie gezeigt wird, fiihren Wechselwirkungen von Allyl-, Butyl-, Benzyl-und Phenyl-ITC mit Eiklarproteinen (frisches Eiklar) zur Bildung von ITC-ProteinKonjugaten. Die ITC reagierenmit Aminogruppen unter Bildung von Thioharnstoff-Derivaten. Diese Reaktionen lassen sich iiber Anderungen (Abnahme) im Gehalt an freien Aminogruppen und an verfiigbarem Lysin gut beschreiben. Die Sulfhydryl-Gruppen der Proteine reagieren ebenfalls mit den ITC unter Bildung von Dithiocarbamidsaureester (Nachweis iiber die Schwefelkohlenstoff-Freisetzung aus diesen Estern). Infolge dieser Reaktionen wird in Abhangigkeit von Art und Menge des eingesetzten Isothiocyanates die pH-abhangige Proteinloslichkeit herabgesetzt, wobei gleichzeitig eine Verschiebung des isoelektrischen Bereiches in Richtung niedrigerer pH-Werte erfolgt. Die elektrophile Reaktion der ITC mit den Proteingruppen fiihrt weiterhin zu einer Veranderung im elektrophoretischen Verhalten (Harnstoff-und SDS-PAGE) der Proteine. Von den untersuchten Isothiocyanaten zeigt sich das Benzyl-ITC als der reaktivste Partner gegenuber Eiklarproteinen.

Benzyl-ITC (benzyl isothiocyanate) reacts preferentially with amino groups and sulfhydryl side chains of bovine sarcoplasmic proteins to form thiourea and dithiocarbamate derivatives, generally resulting in a decrease in solubility of the derivatives along a wide pH range. Under these conditions, it was also possible to show that secondary amine side chains, as found in tryptophan, also react with benzyl-ITC. A quenching of tryptophan fluorescence intensity after interaction with benzyl-ITC was also observed. Polyacrylamide gel electrophoresis (PAGE) experiments in the presence of urea indicated changes in electrophoretical mobility and in composition of subfractions. Changes in surface hydrophobicity and composition as determined by the ANS (8-anilinonaphthalene-1-sulfonate) method and RP-HPLC were also observed. Polymerisation of protein molecules after reaction with benzyl-ITC was documented using the SDS-PAGE technique. These investigations showed that a group of subfractions belonging mainly to glycolytic enzymes and associated proteins with molecular weight between 38-70 kDa was highly reactive.

Glucosinolates and their highly reactive breakdown products (mainly isothiocyanates, ITC) belong to the group of very important natural toxicants. This paper describes the interactions of benzyl-ITC with the egg-white protein fractions ovalbumin, conalbumin, ovomucoid, and lysozyme and the resulting formation of protein-ITC derivatives. Benzyl-ITC reacts with the amino groups of the proteins, forming thiourea derivatives. Such reactions are characterised by decreases in the levels of free amino groups and available lysine. Other reaction sites of benzyl-ITC are sulphydryl side-chains of proteins, the results being formation of dithiocarbamate esters. This in turn results in a decrease of SH-groups due to carbon disulphide being released from their esters. The present study describes the reactions of benzyl-ITC with amino groups and sulphydryl sidechains, and the subsequent decrease of lysine and crysteine after derivation.Changes in the behaviour of ITC-derived egg-white protein fractions were analysed by SDS-PAGE and RP-HPLC.