Glass matrix-facilitated thermal reduction: a tool for probing reactions of met hemoglobin with nitrite and nitric oxide

Abstract

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.

Figure 1

Glass protocols used in the heating experiments.

Figure 2

Heating induced changes in the visible absorption spectrum of aquomet Hb in a single thin glassy film derived from trehalose sucrose (referred to as a G1 glass) by itself (panel a) and sandwiched with a glassy film comprised of trehalose sucrose doped with tagatose (referred to as a G3 glass) (panel b).

Figure 3

Heating induced changes in the visible absorption spectrum of aquomet Hb in a single thin glassy film derived from trehalose sucrose (referred to as a G1 glass) doped with 1 mM sodium nitrite. Panel a-the G1 glass by itself; and Panel b- the G1 glass containing Hb and nitrite sandwiched with a G3 glassy film.

Figure 4

Heating induced changes in the visible absorption spectrum of aquomet Hb in a thin G1 glassy film sandwiched with a G3 thin glassy film containing 10 mM sodium nitrite.

Figure 5

Heating induced changes in the visible absorption spectrum of nitrite met Hb in a thin G1 glassy film containing 0.1 M nitrite sandwiched with a G3 thin glassy film.

Figure 6

Heating induced changes in the visible absorption spectrum of nitrite met Hb in a very dry thin G3 glassy film containing 0.1 M nitrite. Also shown are the progressive heating induced changes to the Soret band in blue region of the spectrum.

Figure 7

Heating induced changes in the visible spectrum of a nitrite met Hb sample in a thin G1 glass film containing 0.1 M nitrite that is linked to a spatially well-separated (2 cm) G3 film through a G1 (trehalose/sucrose) glassy wire.

Figure 8

Heating induced changes in the visible absorption spectrum of aquo met Hb in a G1 glass film that is linked through a G1 glass wire to a spatially well separated source of thermally generated NO.