Second harmonic generation of glucose oxidase at the air/water interface

We present a study of the adsorption of the glucose oxidase enzyme (GOx) at the air/water interface, using the nonlinear optical technique of surface second harmonic generation (SSHG). Resonant SSHG experiments were achieved by probing the pi-pi* transition of the flavin adenine dinucleotide (FAD) c hromophores embedded in the GOx protein. Because of the subsequent resonanc e enhancement of the signal, the second harmonic (SH) wave arising from the GOx entities adsorbed at the interface was detectable for protein bulk aqu eous concentrations as low as 70 nM. The protein adsorption was followed, a nd, at high GOx coverage, a change in the orientation of the FAD chromophor e was observed, indicating either a rearrangement or a reorientation of the protein at the interface. Inasmuch as GOx is negatively charged at the bio logical pH of 7, its interactions with charged surfactants were also invest igated. As expected, spreading positively charged surfactants onto a partia l protein monolayer was found to increase the GOx surface concentration, wh ereas in the case of negatively charged surfactants, the GOx surface concen tration decreased until the SH signal went back to the pure buffer solution response level. With the increasing GOx surface concentration, the rearran gement or reorientation of the protein was also observed.