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Isolating elemental steps that comprise a protein reaction in solution is a difficult process. In this study, the use of sugar-derived glass matrices is evaluated as a biophysical tool to help dissect out elemental steps and isolate intermediates. Two features of the glass are utilized in this endeavor: (i) the capacity of trehalose glass matrices to support thermal reduction over macroscopic distances; and (ii) the ability of glass matrices to significantly damp large amplitude protein dynamics. The focus of the study is on the reaction of nitric oxide (NO) with a nitrite ion coordinated to the heme iron of hemoglobin (Hb). The thermal reduction property of the glass is used to generate NO from nitrite within the glass, and the damping of protein dynamics is used to control entry of NO into the distal heme pocket of Hb, where it can either interact with bound nitrite or bind to the heme iron. The results not only relate to earlier controversial studies addressing the reactions of Hb with NO and nitrite but also raise the prospect that these properties of sugar-derived glassy matrices can be exploited as a new biophysical tool to modulate and probe reactions of NO with hemeproteins as well as a wide range of other metalloproteins.

Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms. This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties. The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-dependent catalytic process whereby Hb generates relatively long-lived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.

A new platform using biocompatible materials is presented for generating powders comprised of nanoparticles that release therapeutic levels of nitric oxide (NO) in a controlled and sustained manner. The capacity of these particles to retain and gradually release NO arises from their having combined features of both glassy matrices and hydrogels. This feature allows both for the generation of NO through the thermal reduction of added nitrite by glucose and for the retention of the generated NO within the dry particles. Exposure of these robust biocompatible nanoparticles to moisture initiates the sustained release of the trapped NO over extended time periods as determined both fluorimetrically and amperometrically. The slow sustained release is in contrast to the much faster release pattern associated with the hydration-initialed NO release in powders derived from glassy matrices. These glasses are prepared using trehalose and sucrose doped with either glucose or tagatose as the source of thermal electrons needed to convert nitrite to gNO. Significantly, the release profiles for the NO in the hydrogel/glass composite materials are found to be an easily tuned parameter that is modulated through the specific additives used in preparing the hydrogel/glass composites. The presented data raise the prospect that these new NO releasing nanoparticles can be easily formulated for use under a wide range of therapeutic circumstances.