In this study we analysed the conversation of Fp-AB with flavin-carrying proteins and investigated the possibility of neutralizing these antibodies by i.v. with dilated cardiomyopathy show a high incidence of serum autoantibodies (M7) [1] directed against mitochondrial flavoproteins (Fp-AB) [2]. these antibodies bind flavins (riboflavin, FMN or FAD) in the nanomolar range, with an apparent kD of 10 nmol. Transport of flavins in the blood takes place predominantly in a protein-bound form, and, besides albumin [3], immunoglobulins represent the predominant class of flavin-binding proteins in the serum [4]. Riboflavin and FAD are the major flavin forms detected in serum, with FMN occurring only in trace amounts. Seventy-two percent of flavins are precipitated with plasma globulins [3], and immunoglobulin subclasses IgG, IgM and IgA have been isolated from serum of normal humans by flavinyl affinity chromatography. The main flavin-binding IgG subclass is usually IgG2 with an apparent kD for riboflavin of 0.23 m [5]. Fab fragments generated by papain digestion of the immunoglobulins also bind riboflavin, indicating that part of the antigenic binding site may be involved [5]. Blood cells contain several times more flavin than serum [4] due to the presence of flavoenzymes in reticulocytes and leucocytes. As shown previously [2], it is possible to isolate Fp-AB from your T-26c serum of patients with myocarditis and dilated cardiomyopathy using affinity chromatography on immobilized FAD-enzyme. No such portion was obtained from the sera of control individuals. Thus the Fp-AB portion was not identical to the flavin-binding portion of immunoglobulins. Its occurrence must reflect the development of an immune response in the patients. In this study we Mouse monoclonal to MDM4 analysed the conversation of Fp-AB with flavin-carrying proteins and investigated the possibility of neutralizing these antibodies by i.v. administration of vitamin B2. Epitope mapping results and the cellular site of Fp-AB-binding decided on both neonatal rat cardiomyocytes and histological section of human heart are discussed. PATIENTS AND METHODS Patients Patients selected for this study showed high titres of Fp-AB. They offered dilated hearts with systolic dysfunction and unexplained heart failure of variable period in the absence of coronary artery or valvular heart disease as documented by heart catheterization, echocardiography, myocardial scintigraphy, and coronarography. Sera from healthy individuals were included in the study as controls. Chemicals Immunochemicals, sarcosine oxidase, riboflavin binding protein from egg white and protein weight markers were obtained from Sigma (Deisenhofen, Germany), 5-bromo-4-chloro-3-indolyl-phosphate, nitrotetrazolium and through a Centricon 10 (Amicon Inc., Beverly, MA) into a protein-free filtrate and a serum protein portion. The flavin content in the fractions was decided according to [11]. Protein digestion 6HDNO (100 g) was digested with trypsin, chymotrypsin and endopeptidase Lys for 4 h at room temperature and T-26c then subjected to SDSCPAGE and to Western blotting. Immunofluorescence microscopy Frozen sections of human, left-ventricular heart tissue and primary cultures of neonatal rat cardiomyocytes [12] were treated with purified Fp-AB from patients, followed by incubation with FITC-labelled rabbit anti-human antibodies and microscopic analysis. RESULTS Conversation of Fp-AB with flavin-carrying proteins The presence of Fp-AB in the serum of patients with heart disease T-26c raises the possibility of an immunological reaction between the flavin-carrying proteins and these antibodies. The subsequent formation of immune complexes in the serum could be detrimental to the patient’s health. We T-26c tested the ability of the affinity-purified Fp-AB from patients’ serum to interact with flavin-carrying serum proteins using immunoelectrophoresis. As shown in Fig. 1, there was no formation of precipitation lines with the Fp-AB. The control with anti-human immunoglobulins, however, gave the expected precipitation lines (Fig. 1). The absence of an immunoreaction between Fp-AB and flavin-carrying serum proteins could reflect the fact that this hydrophobic riboflavin moiety was hidden inside the carrier protein, as it is in the cofactor-binding pocket of flavoenzymes [2], and was therefore inaccessible to the antibodies. Open in a separate windows Fig. 1 Conversation of Fp-AB with serum proteins. Serum proteins were separated electrophoretically under native conditions on agarose; affinity-purified human Fp-AB and as control anti-human IgG, IgA, IgM were then applied to the agarose as indicated and the formation of precipitation lines was monitored. To test this assumption.