Rapid determination of binding parameters of chitin binding domains using chitin-coated quartz crystal microbalance...

Chitin present in fungal cell walls has been considered as a diagnostic polymer for the detection of fungal infections. Chitin staining can be achieved with different dyes such as Calcofluor white or Congo red, but these methods have not entered into clinical routine diagnosis due to problems with sensitivity and specificity. More accurate detection can be achieved using chitin binding domains (CBDs) from a large variety of naturally occurring proteins that specifically interact with chitin. The chitin binding properties of most of these proteins have not yet been determined, because chitin is an insoluble fibrillar material rendering accurate determination of chitin binding kinetics challenging. Here we report a quartz crystal microbalance with dissipation monitoring (QCM-D) based method to determine binding constants of CBDs on chitin-coated gold surfaces. For this purpose, chitin was trimethylsilylated and coated onto the sensor chips. After desilylation, regular fibril-like structures with a typical center-to-center spacing of 85 nm were observed by atomic force microscopy. Using different experimental conditions and data evaluation methods for QCM-D measurements, we determined kon and koff and calculated the KD values for binding of a recombinant CBD from Bacillus circulans chitinase A1. Depending on the evaluation method, the KD values ranged between 0.6 and 2.5 μM. The obtained KD values were in good agreement with those measured for other bacterial CBDs usually ranging between 1 to 10 μM. Hence, we propose that the experimental approach developed in this study can be applied to determine yet unknown binding affinities of various CBDs from different origin.