Spontaneous methylation of hemoglobin by S-adenosyl-methionine by a specific and saturable mechanism.

The methyl group from S-adenosylmethionine (AdoMet) is transferred into hemoglobin without any evident involvement of an enzyme. There are multiple sites for incorporation of the methyl group into hemoglobin, since both alpha and beta chains are methylated. The methyl linkages formed in hemoglobin are stable at both alkaline and acidic pH, and the reaction occurs optimally at slightly below neutral pH. Only a small fraction (approximately 2%) of hemoglobin tetramers are methylated under the conditions tested. Acid hydrolysis of [3H-methyl]-labeled hemoglobin and determination of phenylisothiocynate derivatives yields N epsilon-methyl lysine, which accounts for about one-half of the incorporated [3H-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. Methyl group transfer to hemoglobin from AdoMet is slow and inefficient (kcat/Km approximately 5 x 10(-2), but the reaction velocity tends toward a plateau with increasing AdoMet concentration in a manner suggesting that saturable binding of AdoMet onto hemoglobin is involved in methyl transfer. The velocity of hemoglobin methylation is inhibited by S-adenosylhomocysteine, the known end-product inhibitor of methyltransferases, a further indication that methyl group transfer involves binding and catalysis by a specific site (or sites) in the hemoglobin molecule. These observations may help to explain the known existence of methylated hemoglobins in erythrocyte.