Download CARBOXYL GROUPS The δ- and ε-carboxyl

From: Chemical Modification of Proteins. Gary E. Means and Robert E. Feeney. Holden-Day,
Inc. San Francisco, 1971 pp. 222-223
CARBOXYL GROUPS
The δ- and ε-carboxyl groups of aspartic and glutamic acids, respectively, are the
principal anionic groups in proteins. They are acidic groups with pK values usually
between pH 4.5 and 5.0. They can be esterified under relatively mild conditions by
reaction with one of several diazoacetate derivatives (see Section 7-1). However, only a
relatively small number of the most reactive groups become modified. A harsher
procedure giving more complete reaction requires placing the protein in anhydrous
methanol containing a moderate amount of hydrochloric acid. The following procedure
is essentially that described by Fraenkel-Conrat and Olcott (1945).
The dry protein is suspended in cold absolute methanol and concentrated
hydrochloric acid or hydrogen chloride gas is added to a final acid concentration of 0.02
to 0.10 N. The reaction mixture is maintained between 0° and room temperature for one
to several days. The exact time, temperature, and acid concentration may be varied
depending upon the extent of esterification desired and will usually have to be chosen on
the basis of several trial preparations. At 2° using 0.07 N HCl, for example, nearly 300
hours is required to completely esterify pancreatic ribonuclease whereas eight of its
eleven carboxyl groups are modified in about 120 hours. With most proteins using 0.1 N
HCl and a temperature of 25° reaction will be completed within 24 hours. Reaction is
stopped finally by dilution with a large volume of ice-cold water, and the excess acid and
methanol are removed by dialysis against 0.001 N HCl. Dialysis against neutral or
slightly alkaline solutions results in slow saponification of the methoxyl groups. In
moderate to strongly alkaline solution, or in the presence of hydroxylamine,
saponification is quite rapid and can be used to regenerate the unmodified protein.
Saponification of ribonuclease methyl ester at pH 10.4 and 26 hours (Broomfield et al.,
1965).
Estimation of the extent of the reaction is possible by comparison of the modified
and unmodified proteins’ titration curves between pH 2.0 and ~7.5. The presence of socalled buried or abnormally titrating carboxyl groups in some proteins can be a source of
error. The extent of reaction can be determined based on the amount of methanol
released during alkaline hydrolysis using distillation to separate it from the hydrolysate
and then effecting its oxidation with excess dichromate and backtitrating with ferrous
sulfate (Vithaayathil and Richards, 1961). A third method involves reduction of the
methoxyl groups with lithium borohydride in tetrahydrofurane and determination of the
resulting hydroxyamino acid residues (Chibnall et al., 1958; Vithayathil and Richards,
1961).
Carboxyl groups of proteins can also be converted to amides through reaction
with one of several amines promoted by a water-soluble carbodiimide (Section 7-2). For
quantitative reaction, urea, guanidine/HCl, or another denaturant, can be used as in the
following procedure of Hoare and Koshland (1967) for the quantitative determination of
protein carboxyl groups. The same procedure, but in the absence of a denaturing agent,
can be used to selectively modify proteins affecting up to about half the carboxyl groups.
In a typical reaction, a solution at pH 4.75 containing the protein (13.3 mg/ml),
1.33 M glycine methyl ester, and 7.5 M urea is kept at 25° in a water-jacketed vessel
attached to a pH-stat. A solution of 0.40 M carbodiimide [i.e., l-benzyl-3-(3dimethylaminopropyl) carbodiimide or l-ethyl-3-(3-dimethylaminopropyl) carbodiimide]
in 7.5 M urea is then added to a concentration of reagent of 0.1 M, and the pH is
maintained by automatic titration with 0.5 M HCl. After standing at 25°, the solution is
dialyzed at 0° against 0.001 M HCl. The number of groups modified can be determined
by amino acid analysis after acid hydrolysis to detect the increase in the amount of
glycine. For proteins having a high glycine content, other amines or radioactive glycine
methyl ester can be used. Different amines can also be used to give the modified groups
particularly desired properties. Thus, by using amino-ethanesulfonic acid the negative
charge of the original carboxyl group can be maintained or, conversely, by using a
diamine such as ethylenediamine, it can be replaced by a positive charge.
REFERENCES
Fraenkel-Conrat, H., and H.S. Olcott (1945): J. Biol. Chem., 161, 259.
Broomfield, C.A., J.P. Riehm, and H.A. Scheraga (1965): Biochemistry, 4, 751.
Vithayathil, P.J., and F.M. Richards (1961): J. Biol. Chem., 236, 1380.
Chibnall, A.C., J. L. Mangan, and M.W. Rees (1958): Biochem. J., 68, 114.
Hoare, D.G., and D.E. Koshland (1967): J. Biol. Chem., 242, 2447.