129Xe NMR biosensors are promising agents for the early detection of diseases, given that biomolecular interactions can perturb 129Xe chemical shifts well beyond the typical field inhomogeneity of clinical MRI. We introduce human carbonic anhydrase (CA) as a single-binding-site enzyme for studying xenon biosensor-protein interactions. A xenon-binding cryptophane was substituted with linkers of varying lengths to p-benzenesulfonamide to yield non-diastereomeric biosensors with a single 129Xe NMR resonance. X-ray crystallography confirmed binding of the 8-bond-linked biosensor containing a single xenon atom in the CAII active site. Biosensor dissociation constants (Kd = 20–110 nM) were determined by isothermal titration calorimetry (ITC) for isozymes CA I and II. The biosensor-CA complexes yielded “bound” hyperpolarized 129Xe NMR resonances of narrow linewidth that were shifted by 3.0 to 7.5 ppm downfield, signifying much larger shifts than seen previously. Moreover, isozyme-specific chemical shifts clearly differentiated CA I and II, despite their similar active-site architectures. Thus, xenon biosensors may provide a powerful strategy for diagnosing human diseases characterized by the upregulation of specific CA isozymes and other protein biomarkers.

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