Studies on the radiohalogenation of proteins using the enzymes bromoperoxidase and myeloperoxidase

Three halogenating enzymes - bromoperoxidases isolated from the red algae Bonnemaisonia hamifera or Penicillus capitatus (BPO) and myeloperoxidase (MPO) isolate from canine leukocytes - were used to catalyze the radiohalogenation of proteins at neutral pH. Human serum albumin and canine fibrinogen were halogenated as model compounds; the proteins were labeled with 77Br. For each enzyme, the essential reaction parameters (including the concentration of hydrogen peroxide, the concentration of protein, the amount of enzyme used to catalyze the reaction, the pH of the reaction mixture, and the reaction time) were varied to obtain those conditions which resulted in the highest yield of radiolabeled protein. The data obtained from reaction parameter variation studies for the radiobrominations catalyzed by BPO (P. capitatus) and MPO were similar to those for other halogenating enyzmes; similar studies with BPO (B. hamifera), however, gave anomalous results. These discrepancies are attributed to the presence of carrier bromine in the crude BPO (B. hamifera) enzyme extract. The sites of attachment of the radiobromine to proteins labeled using these three enzymes were investigated. In all cases, the labeled proteins contained bromotyrosyl residues as the predominantly labeled species. The integrity of proteins labeled with bromine-77 using these enzymatic techniques was investigated by several in vivo and in vitro methods, including in vitro hydrolysis, in vitro clottability, in vivo clearance, and thrombus uptake. In all cases, the 77Br-proteins wre superior to proteins radioiodinated by conventional methods, indicating that the enzymatic labeling methods result in stable attachment of the radiobromine to the protein molecules without significantly altering the structure of the protein