Water-dispersed, spherical, underivatized Au-NPs with particle size less than 5 nm were synthesized from an aqueous solution of 32-macrocyclic polyammonium chloride, [32]ane-(NH2+)(8)center dot 8Cl(-) (32-MCPAC) using sodium borohydride (NaBH4) as the reducing agent. The bioconjugation of the synthesized Au-NPs at different pHs (3.6-5.6) with bovine serum albumin (BSA) protein was studied using UV-Vis, fluorescence, and Raman spectroscopy. These studies support that the Au-NPs were incorporated into the protein moiety and bound to it chemically. The binding constants (K-b) and stoichiometries (n) (i.e., the number of Au-NPs bound by the proteins) of BSA protein to the Au-NPs at different pHs were determined by measuring the quenching of the fluorescence intensity of the tryptophan residues of the protein molecules after conjugation. The values for K-b (n) were found to be 1.05 x 10(10) M-1 (1.66), 2.09 x 10(10) M-1 (2.30), and 1.86 x 10(10) M-1 (1.75) at pH 3.60, 4.60, and 5.60 for BSA-Au-NPs conjugations, respectively. The results show that BSA binds to the Au-NPs strongly at pH 4.60, which is equivalent to its isoelectric point (pl 4.6). (C) 2009 Elsevier Inc. All rights reserved.