Using capillary electrophoresis to follow the acetylation of the amino groups of insulin and to estimate their basicities

Abstract

Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein--insulin and its acylated derivatives--as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G alpha and F alpha) and one epsilon-amino group (K epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (G alpha) = 8.4; pKa (F alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD > or = 6 x 10(3) M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.

Equilibrium binding model of bile salt-mediated chiral micellar electrokinetic capillary chromatography

“…The experimental mobility change of insulin during dimerization is in accordance with that predicted from the Offord equation [28]. In a further complexation study, Offord parameters were determined using compounds of known charge and molecular mass to approximate the mobility of the vancomycin-dansyl-valine complex, whose quantity was not accessible experimentally with sufficient precision [29].…”

Protein Charge Ladders, Capillary Electrophoresis, and the Role of Electrostatics in Biomolecular Recognition

“…Charge ladders in combination with CE have been used to estimate the charge of the unmodified protein 16 and the values of pK a of individual ionizable groups. 17,18 Charge ladders in combination with affinity capillary electrophoresis (ACE) make it possible to quantify the contributions of electrostatics to the free energies of binding of charged ligands to proteins. 19 Although we have developed this approach primarily using charge ladders of bovine carbonic anhydrase II (BCA II, EC 4.2.1.1.)…”

“…as a model system (Figure 1), 16,19 we have demonstrated the formation of charge ladders with a range of other proteins and biological molecules. 17,18,[20][21][22][23] …”

Warum sind Proteine geladen? Netzwerke aus Ladungs‐Ladungs‐Wechselwirkungen in Proteinen, analysiert über Ladungsleitern und Kapillarelektrophorese

“…Zudem ist ein Sortiment an gut charakterisierten Inhibitoren für CA (fast alle Arylsulfonamide) erhältlich oder einfach zu synthetisieren. [132] Bei Untersuchungen mit Ladungsleitern haben wir auch Insulin, [133] Lysozym [107,134] und a-Lactalbumin [56] eingesetzt. Außer diesen bilden viele andere Proteine gut aufgelöste Ladungsleitern, [54] allerdings haben wir ihre elektrostatischen Eigenschaften nicht mit dieser Technik analysiert.…”

“…[133] Bei der Herstellung von Ladungsleitern durch Acylierung der Lysinreste werden gewöhnlich 5-25 ¾quivalente eines Anhydrids oder eines NHS-Esters (relativ zum Protein) zur Lösung eines Proteins in 10 Vol.-% 0.1n NaOH (pH % 10) zugegeben. [54,133] [137] durchgeführt werden, diese dürfen aber nur schwach nucleophil sein, damit die Reaktion mit dem Puffer nicht mit der Acylierung konkurriert. Auch Carbamylierungen mit Kaliumcyanat können eingesetzt werden, um Lysinreste durch Überführung in Homocitrullin zu neutralisieren.…”