Isolation of intermediate compounds between hemoglobin and carbon monoxide

M. Perrella, L. Benazzi, L. Cremonesi, S. Vesely, G. Viggiano, L. Rossi-Bernardi

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A human hemoglobin solution partially saturated with carbon monoxide was rapidly quenched at -25°C into a hydro-organic buffer containing ferricyanide. Under the experimental conditions of pH, ionic strength, and buffer composition used in this work, it was found that the deoxy hemes were rapidly transformed into their met form, whereas practically no carbon monoxide-bound hemes were oxidized before the separation of the mixture from the oxidizing agent. As a preliminary step to the analysis of the resulting solution carbonylhemoglobin solutions partially oxidized with ferricyanide were studied by isoelectric focusing at -25°C under identical conditions. The relative position in the gel of all nine possible valence hybrids was established as follows (going from the anodic to the cathodic side of the gel) α2(CO)β2(CO), (α(CO)β+)(α(CO)β(CO)), (α2(CO)β2+), (α+β(CO))(α(CO)β(CO)), (α+β+)-(α(CO)β(CO)), (α+β+)(α(CO)β+), (α2+β2(CO)), (α+β+)(α+β(CO)), α2+β2+. When carbonylhemoglobin and methemoglobin were mixed in equal proportion at -25°C and then analyzed by isolecteric focusing at the same temperature, it was found that the contribution of valence of hybrids other than α2(CO)β2(CO) and α2+β2+ to the total amount of hemoglobin in the gel was no more than 6%. When carbonylhemoglobin and deoxyhemoglobin were mixed in the same proportion and incubated at 20°C so to allow the redistribution of the carbon monoxide molecules between all possible binding sites to occur, a substantially higher amount of valence hybrids, derived from the oxidation of intermediate compounds of hemoglobin with carbon monoxide, was found. The isoelectric focusing separation indicated the presence in the original solution of intermediate species other than carbonylhemoglobin and deoxyhemoglobin at a concentration of about 10% of the total.

Original languageEnglish
Pages (from-to)4511-4517
Number of pages7
JournalJournal of Biological Chemistry
Issue number7
Publication statusPublished - 1983

ASJC Scopus subject areas

  • Biochemistry


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