Effects induced by hydroxyl radicals on salmon calcitonin: a RP-HPLC, CD and TEM study Maria Cristina Gaudiano a , Marco Diociaiuti b, * , Paola Bertocchi a , Luisa Valvo a a Dipartimento del Farmaco, Istituto Superiore di Sanita `, Viale Regina Elena 299, 00161 Rome, Italy b Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanita `, Viale Regina Elena 299, 00161 Rome, Italy Received 4 July 2002; received in revised form 23 July 2003; accepted 25 July 2003 Abstract The effects of hydroxyl radical attack on a peptidic drug were studied in vitro. Different chemico-physical techniques were used to investigate structural damage induced by oxidative stress conditions in salmon calcitonin (sCT), a peptide hormone used in treating osteoporosis. Reversed-phase liquid chromatography (RP-HPLC), circular dichroism (CD) and transmission electron microscopy (TEM) were applied to measure formation of oxidation/degradation products and to reveal the conformational and ultrastructural modifications in the presence of OHÁ free radicals. Hydroxyl radicals were obtained from ferrous sulfate and ascorbic acid mixtures. The RP-HPLC results revealed the formation of new chromatographic peaks indicating a number of degradation/oxidation products formed in the presence of OHÁ free radicals. CD spectra showed slight protein conformational modifications as well as aggregation. TEM confirmed sCT aggregation and suggested the formation of fibrillar aggregates. D 2003 Published by Elsevier B.V. Keywords: Oxidative stress; Hydroxyl radicals; Salmon calcitonin; RP-HPLC; CD; TEM 1. Introduction Overproduction of hydroxyl radicals in vivo is a physi- ological condition during aging and in such pathologies as strokes, rheumatoid arthritis, cancer, Alzheimer’s and Par- kinson’s diseases [1–5]. Under these conditions, the prima- ry structure of endogenous and exogenous peptides and proteins could be modified leading to the formation of aggregates and fibrils [6]. This process has been described for the h-amyloid (hA) peptide, the central constituent of senile plaques in Alzheimer’s disease. In this case, Met35 oxidation to sulfoxide enhances the aggregation rate com- pared to non-oxidized peptide [7]. In some cases, these peptide fibrils explicate cellular toxicity through an oxida- tive mechanism via a free radical pathway [8]. Schubert et al. [9] showed that aggregates of hA, amylin and human calcitonin from fibrillar deposits of medullary carcinomas were toxic to B12 cells. The authors proposed that the toxicity was due to free radical-induced lipid peroxidation. Other authors reported that hA peptide is probably the source of free radical-mediated oxidative stress leading to neurodegeneration. The scientific literature increasingly indicates that free radicals can induce polypeptide aggrega- tion by oxidative stress and it now seems evident that some peptide fibrils can induce oxidative reactions causing cell damage and toxicity [7,9]. However, the role of amyloid h protein aggregates as either mediators or by-products of pathogenic processes leading to Alzheimer’s disease is still debated. Bucciantini et al. [10] recently proposed that a common mechanism is involved in protein aggregate toxicity. They demonstrated that even aggregates of non-disease-associat- ed proteins, very similar to amyloid fibrils and plaques, could be inherently highly toxic. The authors concluded that avoiding protein aggregation is crucial for the preservation of biological function and suggested common features in the origin of this family of protein deposition diseases. In the present paper, the effect of OHÁ free radicals on salmon calcitonin (sCT) was studied in vitro to understand if radicalic oxidative reactions affect the degradation process of peptide in solution as well as its conformation and 0304-4165/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/S0304-4165(03)00158-2 * Corresponding author. Tel.: +39-06-4990-2236; fax: +39-06-4938- 7140. E-mail address: marco.diociaiuti@iss.it (M. Diociaiuti). www.bba-direct.com Biochimica et Biophysica Acta 1623 (2003) 33 – 40