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. 2004 Jul 16;101(31):11477–11482. doi: 10.1073/pnas.0402201101

Fig. 3.

Fig. 3.

Determining the source of NO consumption in human plasma. (A) Scheme of experiments performed to determine the source of NO consumption in normal human plasma. HSA, human serum albumin; Hb, hemoglobin; Hp, haptoglobin; IP, immunoprecipitation. (B) Mean relative (%) NO consumption (n = 2-6) by 50-μl injections of the indicated samples. Error bars indicate SEM. *, P < 0.05. (C) Immunoblot of IEF fractions by using antihemoglobin antibodies. A standard curve of hemoglobin was run together with IEF fractions 6-9. (D) Immunoblot of plasma by using antihemoglobin antibodies. (E) Plot of NO scavenging measured by chemiluminescence versus heme concentration measured by hemoglobin ELISA. (F) Immunoprecipitation with antihemoglobin and antihaptoglobin antibodies eliminated plasma NO consumption. (Upper) Immunoblot of plasma and plasma pass-through from the antibody columns. P, plasma pass-through; E, acid glycine eluate from bead-antibody complex. (Lower) NO consumption of plasma and plasma pass-through from immunoprecipitation experiments. (G) Immunoblot with antihemoglobin antibodies showed that hemoglobin was complexed with haptoglobin and migrated slowly as a large-molecular-mass multimeric complex. MW, molecular mass.