Studies on the amide and C-terminal residues in proteins. 3. The esterification of proteins

Abstract

The carboxyl groups of a protein can be esterified with diazomethane, but more conveniently with methanolic hydrochloric acid. In the present research, however, neither reagent has proved to be entirely satisfactory, for in our hands the first has not given full esterification and the second would do this only at the expense of the amide N, which we were particularly anxious to keep intact. Fraenkel-Conrat & Olcott (1945) were the first to show that the concentration of mineral acid (0-02 to 01 N) required to catalyse the reaction with methanol was very much less than previous workers had employed for the purpose. They claimed that 97 % of the carboxyl groups of polyglutamic acid from Bacillu8 8ubtili8 could be esterifled by treatment with methanolic 0-05N-HCI at 22-24°for 24 hr. and that several proteins, including insulin, were fully esterified under similar conditions in the presence of 01IN-HCI. They mentioned that there was no loss of amide N under such treatment, though data in support of the assertion were not presented. Mommaerts & Neurath (1950) repeated the experiments with insulin, and confirmed that full esterification was apparently achieved with methanolic 0-1 N-HCI at 250 in 24 hr. They claimed that the ammonia liberated under these conditions amounted to not more than 2 % of the amide N.Our own observations do not support the contention that 0 1 N-HCI can be used as a catalyst in the esterification of proteins in this way without loss of amide groups. With insulin the ammonia produced in 24 hr. at 25°represents about 6-6 % of the amide N, a value much greater than the abovementioned workers seem to have suspected. Lowering the concentration of hydrochloric acid spares the amide N but unfortunately full esterification is not then achieved. We have thus been obliged to prepare the ester with methanolic 0 1 N-HCI, knowing that when this is used subsequently to determine inter alia the amide distribution of the protein, we had already set a limit to the accuracy with which this could be done. Another interesting side-reaction of protein esterification has been traced to N-O acyl migration in serine and particularly threonine residues. EXPERIMENTAL MateriakProtein samples, and methods for determining N, ammonia N and amide N were described in the preceding paper (Chibnall, Mangan & Rees, 1958b). Methoxyl. The Zeisel procedure of Pregl (1937) was used. Estimations on 20-30 mg. samples agreed to within 2%.Methanol. This was boiled under reflux for some hours with Mg turnings and then distilled.Methanolic hydrochloric acid. An approx. 2N-solution was prepared with dry HCI gas and stored at -150. As the acidity falls on keeping it is necessary to titrate a sample immediately before use. MethodsEstimation of the extent of esteriftcation. The extent of esterification of the total free carboxyl groups was followed by determination of methoxyl. As ,-lactoglobulin contains methionine, which yields methyl iodide on treatment with HI, the (apparent) methoxyl content of the protein itself was subtracted from that...

Studies on the interaction of magnesium, calcium and strontium ions with native and chemically modified human serum albumin

“…Native human albumin was prepared from plasma by the cold-ether fractionation technique of Kekwick & Mackay (1954). Chemically modified albumins were prepared by standard procedures as follows: esterification, by the method of Chibnall, Mangan & Rees (1958); acetylation, by the method of Marrack & Orlans (1954); bromoacetylation, by the method of Korman & Clarke (1956); phosphorylation, by the method of Ferrel, Olcott & Fraenkel-Conrat (1948).…”

“…Amide groups were determined by the method of Chibnall et al (1958), and free amino groups by the method of Baddiley, Kekwick & Thain (1952). Methoxyl groups were measured by the Zeissel method (Grant, 1951) and phosphorus by the method of Allen (1940) after correction for inorganic orthophosphate (Lowry & Lopez, 1946 (2) albumin (2) pH 1-6 2-5 2-3 3-6 4-6 6-6 7-7 8-3 (Orgel, 1958).…”

Peptidyl‐Donor Substrates for Ribosomal Peptidyl Transferase

“…The residue was dissolved in anhydrous trifluoroacetic acid (0.5 ml) and kept for 2 min at 20 "C. The A sample of CpCpA-Leu was hydrolysed with 0.2 M NaOH (5 min, 60 "C) and, after neutralization with 0.2 M acetic acid, the amount of leucine was estimated [28]. On the basis of a millimolar absorption coefficient of 28.1 for CpCpA a t 268 nm and pH 2, the ratio of CpCpA: Leu was found to be 1 .lo : 1 .oo.…”

C-Terminal Labeling of Immunoglobulin G with a Cysteine Derivative by Carboxypeptidase Y Catalyzed Transpeptidation

“…It was found that significant degradation of the IgG was observed by SDS-polyacrylamide gel electrophoresis analysis, even at HCl concentrations as low as 0.05 M and at 25°C for 24 h. However, by employing a harsher procedure, i.e., performing the reaction for 48 h at the lower temperature of 4°C in the presence of 0.2 M HCl, degradation was no longer apparent by electrophoretic analyses, as shown in . The change in the mobility of the protein probably results from charge changes in the carboxyl functions of glutamic acid, aspartic acid, or C-terminal residues by methyl ester formation and methanolysis of the amide functions of glutamine and asparagine residues due to side reactions (21). Evidence from electrophoresis confirms that the integrity of the IgG molecule is maintained during the process of methyl esterification.…”