J. Biol. Chem., Vol. 260, Issue 10, 6039-6046, 05, 1985
Selectivity in the modification of the alpha-amino groups of hemoglobin on reductive alkylation with aliphatic carbonyl compounds. Influence of derivatization on the polymerization of hemoglobin S
AS Acharya, LG Sussman and JM Manning
The reactivity of the alpha-amino groups of the alpha- and beta-chains of
hemoglobn toward reductive alkylation using limiting concentrations of the
aliphatic carbonyl compounds, acetaldehyde (ethylation), glyoxylic acid
(carboxymethylation), glycolaldehyde (hydroxyethylation), glyceraldehyde
(dihydroxypropylation), and dihydroxyacetone (dihydroxyisopropylation) has
been investigated. Hemoglobin A reductively ethylated at the alpha-amino
groups eluted on CM-52 ahead of unmodified hemoglobin A, and hemoglobin A
reductively ethylated at the epsilon-amino groups. This observation is
similar to that seen on hydroxyethylation and dihydroxypropylation of the
alpha- amino group of hemoglobin A. The presence of the alpha-hydroxyl or
the carboxyl group in the carbonyl component used in the reductive
alkylation influences considerably the selectivity pattern during the
derivatization. The alpha-amino groups of the alpha- and beta-chains are
modified to nearly the same degree during reductive hydroxyethylation as
well as during reductive dihydroxypropylation. Reductive ethylation
(aldehyde lacking the alpha-hydroxyl group) exhibited a slight preferential
reaction at Val-1(beta). The presence of a negatively charged carboxyl
group in the carbonyl component, i.e. glyoxylic acid, made this
preferential reaction at Val-1(beta) even more pronounced. When the
reductive alkylation is carried out with dihydroxyacetone (a ketone instead
of an aldehyde), the dihydroxyisopropylation occurred at a slower rate and
exclusively at Val-1(beta). The ethylation, hydroxyethylation,
carboxymethylation, and dihydroxypropylation of the alpha-amino groups of
hemoglobin S increased its solubility from the value of 16 g/dl for the
unmodified protein to about 25 g/dl for the modified protein. Thus, the
alkyl chains on the alpha-amino groups on the polymerization have a strong
inhibitory influence. In order to determine the influence of the alkyl
chains at the alpha-amino groups of alpha- and beta-chains on
polymerization, hybrid hemoglobin S tetramers with hydroxyethylation either
at Val-1(alpha) or at Val-1(beta) have been prepared. The solubility of
each hybrid is about 26 g/dl. Thus, the hydroxyethyl group either on the
alpha- or the beta-chain appears to interfere with the polymerization of
deoxygenated HbS to the same degree. The inhibitory influence of the
hydroxyethyl chain at Val-1(alpha) on the polymerization, compared with the
lack of such an influence when this alpha-amino group is modified by
cyanate, suggests that a carbamoyl group on Val-1(alpha) can be
accommodated in the intermolecular contact region involving this segment of
the molecule without seriously perturbing the mo
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