Binding of Heme-CO to Bovine and Porcine β-Lactoglobulins

Two molecules of heme-CO bind to bovine or porcine β-lactoglobulin (BLG) with an average affinity of 0.5 μM. The affinity increases with pH, with a transition near pH 7.5, indicating a conformational change in the protein. Heme-CO does not bind to the predominantly α-helix conformation of BLG, which occurs in solutions with more than 40% ethanol or methanol. Fluorescence energy transfer measurements have been made for the complex of BLG with retinol and/or heme-CO. Two species of BLG were used. While bovine BLG possesses two tryptophans (at positions 19 and 61) which are quenched by about a factor of 2 by either retinol or heme-CO, the porcine species has only one tryptophan (at position 19) whose fluorescence is decreased by a factor of 15 when both hemes are bound, indicating that at least one of the heme-binding sites is near (<20 Å) to this tryptophan. The fluorescence of retinol (complexed to BLG) is also quenched by the addition of heme-CO, indicating that BLG can bind both molecules simultaneously; a separation of 25 Å between retinol and heme was calculated. The results suggest at least two hydrophobic pockets for this protein.