[Frontiers in Bioscience 10, 838-843, January 1, 2005]

REACTIVE OXYGEN SPECIES (ROS) INDUCE CHEMICAL AND STRUCTURAL CHANGES ON HUMAN INSULIN IN VITRO, INCLUDING ALTERATIONS IN ITS IMMUNOREACTIVITY

Ivonne M. Olivares-Corichi 1, Guillermo Ceballos 2, Clara Ortega-Camarillo 3, Alberto M. Guzman-Grenfell 4, and Juan J. Hicks 4

1Laboratorio de Bioquimica, Direccion de Investigacion y Enseñanza del Hospital Juarez de Mexico, D.F. Mexico, 2Laboratorio Multidisciplinario de Investigacion, Seccion de Graduados, Escuela Superior de Medicina del Instituto Politecnico Nacional, D.F. Mexico, 3Unidad de Investigacion Medica en Bioquimica del Hospital de Especialidades del Centro Medico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, D.F. Mexico, 4Laboratorio de Bioquimica Inorganica, Unidad de Investigación Instituto Nacional de Enfermedades Respiratorias, Subdireccion de Investigacion Biomedica. D.F. Mexico

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents
3.2. Experimental design
3.3. Hydroxyl (HO), radical generation
3.4. Absorption changes (280 nm) and nitroblue tetrazolium (NBT) reduction
3.5. Protein carbonyl groups
3.6. Radioimmunoassay (RIA)
3.7. Statistics
4. Results
4.1. Absorption changes at 280 nm
4.2. Nitroblue tetrazolium (NBT) reduction
4.3. Protein carbonyl groups
4.4. Radioimmunoassay (RIA)
5. Discusión
6. Referentes

1. ABSTRACT

Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the endogenous antioxidant defense. Peroxidations induced by ROS are the key of chemical and structural modifications of biomolecules including circulating proteins. To elucidate the effect of ROS on circulating proteins and considering the presence of oxidative stress in Diabetes Mellitus, the effects of ROS, in vitro, on human insulin were studied. We utilized the Fenton reaction for free hydroxyl radical (HO) generation in presence of human recombinant insulin measuring chemical changes on its molecular structure. The induced changes in insulin were: a) significant increase on absorbance (280 nm) due to phenylalanine hydroxylation (0.023 +/- 0.007 to 0.13 +/- 0.07). b) Peroxidation products formed on amino acids side branches (peroxyl and alcohoxyl group); measured as increased capacity of reduce nitroblue of tetrazolium (NBT) to formazan (0.007 +/- 0.007 to 0.06 +/- 0.02). c) Increased concentration of free carbonyl groups (8.8 +/- 8.7 to 45.6 +/- 20.2 pmoles dinitrophenylhidrazones/nmol insulin) with lost of secondary structure, and d) Modification of epithopes decreasing the insulin antigen-antibody reactivity measured as a decrease in insulin concentration by RIA. In conclusion, the radical hydroxyl in vitro is able to induce molecular modifications on insulin.