Biochimica et Biophysica Acta, 303 (I973) 44-51 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36339 KINETICS OF THE REACTION OF THE MASKED SULPHYDRYL GROUPS OF HAEMOGLOBINS A, E AND NEW YORK WITH p-CHLOROMERCURIBENZOATE Y. YUTHAVONG* AND P. RUENWONGSA Department of Biochemistry, Faculty of Sciences, Mahidol University, Rama VI Road, Banghok (Thailand) (Received July 7th, i972) (Revised manuscript received October i6th, 1972) SUMMARY The order of reaction of the masked (a lO4 and fl 112) sulphydryl groups of human oxyhaemoglobin with p-chloromercuribenzoate was found to be 0.5 with respect to the haemoglobin component. The rate of reaction for haemoglobin E is approximately twice, and for haemoglobin New York slightly greater than, that for haemoglobin A at pH 7.0, 25 °C and 0.6 ionic strength. The effects of pH, ionic strength and temperature on the reaction rates for haemoglobins A and E were compared. It is concluded that the monomers of the haemoglobins are the only major reactive species, and the dimer-monomer dissociation constants are smaller for the normal haemoglobin than for the abnormal haemoglobins E and New York which have amino acid replacements in the contacts which form at5 dimers. INTRODUCTION Of the six sulphydryl groups of human haemoglobin, the two at fl 93 react rapidly and the other four, at a lO4 and fl II2, undergo a slow reaction with p-chloromercuribenzoie acid (PCMB). The slowly reacting, or "masked", sulphydryl groups are all situated in the alfi I contact region. Chiancone et al. 1 suggested that the species responsible for this slow phase of the reaction are both alfi 2 dimers and the monomers of haemoglobin. However, the existence of alfl 2 dimers has yet to be demonstrated in both oxy- and deoxyhaemoglobin2,3. It is possible that the a IO4 and fl II2 sulphydryl groups only of monomers react with PCMB. Haemoglobins E (/3 26, Glu-÷ Lys) and New York (fl 113, Val--~ Glu) both have amino acid replacements in the alfil contact region, which might well affect the Abbreviation: PCMB, p-chloromercuribenzoate. * Present address: Molecular Biology Institute, U.C.L.A., Los Angeles, Calif. 90024, U.S.A.