The Use of Glassy Films and Sol-Gel Matrices to Probe Nitrite Mediated Reactions of Met Hemoglobin

There have been growing indications that under certain conditions hemoglobin (Hb) can undergo nitrite mediated reactions that result in the formation of bioactive forms of nitric oxide (NO) capable of reversing vasoconstriction due to NO scavenging. This process is especially relevant for the design of Hb based blood substitutes that typically cause vasoconstriction when administered. In this presented work the use of both trehalose-derived glassy films and silane derived sol-gel matrices are used to isolate both reactive intermediates and key steps in nitrite-mediated reactions of met Hb. The glassy films allow for controlled production NO within the glass and controlled access of the NO into the distal heme pocket of the met nitrite derivative of Hb. The use of the sol-gel allows for trapping either the T or R state forms of Hb and for facile separation of products (e.g. nitrosothiols such as GSNO) from the Hb containing sol-gel phase. The contributions of added NO and small thiol containing molecules (L-cysteine and glutathione) are exposed. The results are consistent with the formation of a relatively stable intermediate capable of forming S-nitrosothiols such as GSNO. The intermediate has properties consistent with one proposed by Gladwin, Kim-Shapiro1 and coworkers which has the potent nitrosating agent N2O3 coordinated to a ferrous heme.1. Basu, S., et al, Nature Chemical Biology, (2007) vol. 3, p.785.