The values of charge and electrophoretic mobility of a protein are changed upon acylation of its α- and Lys ε-NH3 + groups. Partial acylation of the amino groups of a protein results in a set of derivatives that is often resolved by capillary electrophoresis into a set of distinct peaksthe “rungs” of a protein charge ladderthat differ incrementally in the number of residues modified. Proteins that have values of MW < 50 kD usually form resolved charge ladders when allowed to react with acetic anhydride, while proteins that have values of MW > 50 kD form broad unresolved peaks. Resolved charge ladders of proteins that have values of MW > 50 kD may be formed using acylating agents that introduce several charges upon acylation of each of their Lys ε-NH3 + groups. As an example, l-lactate dehydrogenase (MW = 147 kD) does not form a resolved charge ladder when modified with acetic anhydride. When it is acylated with either 1,2,4-benzenetricarboxylic anhydride, 3, or 1,2,4,5-benzenetetracarboxylic dianhydride, 4, however, it forms charge ladders in which each of the first several pairs of adjacent rungs are separated by approximately 3 or 4 units of charge, respectively. The procedures described in this paper were used to form resolved charge ladders from 25 proteins differing in pI and in MW.

Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.

The values of electrophoretic mobility, μelectro, of bovine carbonic anhydrase II, human carbonic anhydrase II, cytochrome c, lysozyme, superoxide dismutase, ovalbumin, and derivatives of these proteins produced by partial neutralization of Lys ε-NH3 + and/or Asp and Glu carboxyl groups were measured using capillary electrophoresis (CE). For derivatives of these proteins with the lowest overall values of net charge (either positive or negative), the values of μelectro and the values of charge measured by CE, Z CE, demonstrate a linear correlation with the number of charged groups, n, converted to neutral derivatives. For derivatives of these proteins with larger values of net charge, the values of μelectro and Z CE demonstrate a nonlinear correlation with n. Several observations made in this work suggest that shifts in the values of pk a of the ionizable groups on these proteins likely contribute to the observed nonlinear correlation. Debye−Hückel theory was used to calculate values of electrostatic potential at the surface of the derivatives of all six proteins from the measured values of μelectro. These values were plotted against the values of electrostatic potential calculated by assigning a charge to each protein in direct proportion to n. The data for all six proteins fell along a single common curve, regardless of the concentration of monovalent cations in the electrophoresis buffer.

Crude lipase from Candida rugosa (CRL) is a poorly to moderately enantioselective catalyst for the hydrolysis of esters of 2-substituted carboxylic acids such as 2-arylpropanoic acids (E = 4-10) and 2-(ary1oxy)propanoic acids (E = 2-17). Previous workers converted CRL into a high enantioselectivity form using a four-step purification procedure that included a n organic solvent treatment. In this paper we report a simple 2-propanol treatment that converts crude CRL to the high enantioselectivity form. Dissolving crude commercial CRL in 50% 2-propanol followed by dialysis to remove 2-propanol increased the total activity by a factor of 1.2-1.6 and the enantioselectivity toward seven carboxylic acids by a factor of 2.3 to '25. We demonstrated synthetic use of this 2-propanol-treated CRL by resolving 10 g of methyl 2-(4-chlorophenoxy)propionate (3-methyl ester), yielding 2.8 g of product acid (93.1% ee R, a serum cholesterol-lowering compound) and 4.7 g of recovered ester (94.4% ee S ) , corresponding to a n enantiomeric ratio of 100 for the resolution. Recent X-ray crystal structures identified two conformational forms of CRL-open and closed. We suggest that the 2-propanol treatment may increase the activity and enantioselectivity by converting the closed form of CRL to the open form.

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