Labeling of Human Serum Albumin with Stable Isotope of Bromine; an in Vitro Study

Background: Possibility to trace-label albumin with isotopes results in information concerning its synthesis, breakdown, and distribution in the intra and extra cellular spaces. The iodination of albumin is a widespread procedure used in scientific studies. Bromine not only is more reactive and less expensive than iodine, but bonds more easily with many elements. Therefore, it could be a suitable tracer in labeling procedures. The present study was designed to represent a method for labeling human serum albumin (HSA) with stable isotope of bromine. Methods: In the present study, the labeling of HSA by use of stable isotope of bromine (79Br) has been sought through a series of preliminary experiments including iodination of bovine serum albumin (BSA) and iodination and bromination of BSA. The experiments were basically designed according to that of Mc Conahey and Dixon in 1966. All measurements have been obtained by inductive coupled plasma mass spectrometry (ICP/MS). Twenty protein solutions, each having 50 mg HSA dissolved in 10 ml of 0.05 M buffer (pH 7.0) were prepared. A series of calculated amounts of pure bromine was added directly to each sample. Each sample was placed in a crystallizing dish containing crushed ice to keep the reactants cold. After dialysis and final preparation of the samples, the intensities of bromine in the samples were measured. Results: Data indicated the maximum presence of bromine in HSA samples in a ratio of 40 atoms of bromine to each mole of HSA. After dialysis, sample analysis showed that on average, about 65% of the bromine was really bound to the HSA molecules. This finding indicates that about 26 atoms of bromine were bound to each HSA molecule. Data analyzed by simple linear regression method. Results showed that each µg increase in dose leads to 0.002 unit increase in the mean of mole ratio of pure bromine (Br2)/HSA (P=0.001). Conclusion: The present study has unique specifications in that almost all of the labeling procedures of plasma proteins have used other elements rather than bromine and mostly radioactive isotopes instead of stable isotopes. The present paper showed a method for about minimum as well as maximum bromination of HSA (0.05 atoms- 26 atoms), within certain limits of experimental conditions. By this method, one can exactly determine how much bromine should be used to obtain a certain desired mole ratio of Br2/HSA with no, or at least minimal, alteration of protein behavior.