(b) 6HDNO (100 g per assay) was subjected to treatment with trypsin (lane 2), chymotrypsin (lane 3) and endopeptidase Lys (lane 4)

(b) 6HDNO (100 g per assay) was subjected to treatment with trypsin (lane 2), chymotrypsin (lane 3) and endopeptidase Lys (lane 4). and 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 IQ-R 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 IQ-R shown previously [2], it is possible to isolate Fp-AB from the serum of patients with myocarditis and dilated cardiomyopathy using affinity chromatography on immobilized FAD-enzyme. No such fraction was obtained from the sera of control individuals. Thus the Fp-AB fraction was not identical to the flavin-binding fraction of immunoglobulins. Its occurrence must reflect the development of an immune response in the patients. 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. 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 presented dilated hearts with systolic dysfunction and unexplained heart failure of variable duration 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 fraction. Rabbit Polyclonal to HSL (phospho-Ser855/554) The flavin content in the fractions was decided IQ-R according to [11]. Protein digestion 6HDNO (100 g) was digested with trypsin, chymotrypsin and endopeptidase Lys for 4 h at room temperature and then subjected to SDSCPAGE and to Western blotting. Immunofluorescence microscopy Frozen sections of human, left-ventricular heart tissue and primary cultures of neonatal rat IQ-R 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 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 tested the IQ-R 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.