SPONTANEOUS METHYLATION OF HEMOGLOBIN BY S-ADENOSYLMETHIONINE BY A SPECIFIC AND SATURABLE MECHANISM

The methyl group from S-adenosylmethionine (AdoMet) is transferred int o hemoglobin without any evident involvement of an enzyme. There are m ultiple sires for incorporation of the methyl group into hemoglobin, s ince both alpha and beta chains are methylated. The methyl linkages fo rmed in hemoglobin are stable at both alkaline and acidic pH, and the reaction occurs optimally at slightly below neutral pH. Only a small f raction (similar to 2%) of hemoglobin retramers are methylated under t he conditions tested. Acid hydrolysis of [H-3-methyl]-labeled hemoglob in and determination of phenylisothiocynate derivatives yields N epsil on-methyl lysine, which accounts for about one-half of the incorporate d [H-3-methyl] radioactivity. Other amino acids are methylated as well , with much of the remaining radioactivity being distributed among one or more of the side chains of histidine, cysteine, and arginine. Meth yl group transfer to hemoglobin from AdoMet is slow and inefficient (k (cat)/K-m similar to 5 x 10(-2)), but the reaction velocity tends towa rd a plateau with increasing AdoMet concentration in a manner suggesti ng that saturable binding of AdoMet onto hemoglobin is involved in met hyl transfer. The velocity of hemoglobin methylation is inhibited by S -adenosylhomocysteine, the known end-product inhibitor of methyltransf erases, a further indication that methyl group transfer involves bindi ng and catalysis by a specific site (or sites) in the hemoglobin molec ule. These observations may help to explain the known existence of met hylated hemoglobins in erythrocyte.