Modification of Tryptophan Residues in Retinal-binding Protein and Prealbumin with Z-Hydroxy-5-nitrobenzyl Bromide

Human retinol-binding protein (RBP) and prealbumin were labeled with Z-hydroxy&nitrobenzyl bromide (HNB-Br) at pH 5.5 using 20to loo-fold molar excess of the label over tryptophan in each protein. In prealbumin, only 1 to 1.7 tryptophan residues out of 8 were modified under these conditions. This modification did not alter the capacity of prealbumin to bind to retinol-RBP at physiological ionic strength. When native retinol-RBP and apo-RBP were labeled with HNB-Br, 1.1 to 2.1 out of the total of 4 tryptophan residues were modified. This modification did not affect the ability of RBP to bind to retinol, or the ability of apo-RBP to form the retinal-RBP complex upon addition of retinol. The modified retinol-RBP and reconstjtuted apo-RBP, however, did not form a complex with prealbumin at physiological ionic strength. HNB-Br modification of retinol-RBP-prealbumin complex resulted in 0.7 tryptophan residue being modified in each protein. This modification did not affect the properties of either retinal-RBP, prealbumin, or the interaction between the two proteins. It is concluded, therefore, that 1 of the tryptophan residues modified in native and apo-RBP is involved in the binding between prealbumin and retinol-RBP. In addition, both RBP and prealbumin are capable of binding noncovalently to the HNB chromophore under the conditions of the modification used.