TY - JOUR
T1 - Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients
AU - Doctor, Allan
AU - Platt, Ruth
AU - Sheram, Mary Lynn
AU - Eischeid, Anne
AU - McMahon, Timothy
AU - Maxey, Thomas
AU - Doherty, Joseph
AU - Axelrod, Mark
AU - Kline, Jaclyn
AU - Gurka, Matthew
AU - Gow, Andrew
AU - Gaston, Benjamin
PY - 2005/4/19
Y1 - 2005/4/19
N2 - It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.
AB - It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-β93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased ≈20-fold in human septic shock (P = 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.
KW - Nitric oxide
KW - Sepsis
KW - Vascular physiology
UR - http://www.scopus.com/inward/record.url?scp=20244377241&partnerID=8YFLogxK
U2 - 10.1073/pnas.0407490102
DO - 10.1073/pnas.0407490102
M3 - Article
C2 - 15824313
AN - SCOPUS:20244377241
SN - 0027-8424
VL - 102
SP - 5709
EP - 5714
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 16
ER -