Suppression of Insulin Aggregation by Heparin

Abstract

The aggregation of insulin near its isoelectric point and at low ionic strength was suppressed in the presence of heparin. To understand this effect, we used turbidimetry and stopped-flow to study the pH-and ionic strength (I)-dependence of the aggregation of heparin-free insulin. The results supported the role of interprotein electrostatic interactions, contrary to the commonly held view that such forces are minimized at pH ) pI. Electrostatic modeling of insulin (DelPhi) revealed that attractive interactions arise from the marked charge anisotropy of insulin near pI. We show how screening of the interprotein attractions by added salt lead to maximum aggregation near I ) 0.01 M, corresponding to a Debye length nearly equal to the diameter of the insulin dimer, consistent with a dipole-like protein charge distribution. This analysis is also consistent with suppression of aggregation by heparin, a strong polyanion that by binding to the positive domain of one protein, inhibits its interaction with the negative domain of another.

External Factors Affecting Protein Aggregation

“…Several globular proteins have maximum free energy change ∆ G unf (8 -17 kcal/mol) in a temperature range between 10 and 40 ° C, including basic pancreatic trypsin inhibitor, CT, cytochrome c, lysozyme, metmyoglobin, RNase A, and trypsin. 99 Therefore, some proteins could aggregate easily at room temperature, such as insulin 100 and a therapeutic immunoglobulin. 101…”

“…Recent results suggest that signifi cant protein -protein interactions can still exist from the marked charge anisotropy of protein around its pI, such as insulin. 100 Nevertheless, the rate and extent of protein aggregation should be maximal around the pI and should be negatively correlated with the absolute value of charge. 151,152 Indeed, Militello et al 29 found that the aggregation of BSA increased as pD is adjusted closer to its pI (about 5) at 58 ° C. Giger et al 100 studied aggregation of insulin in the pH range of 3 -9 and found that aggregation (turbidity) of insulin at room temperature was fastest at pH 5.6 (pI = 5.5) in a 10 mM NaCl solution.…”

“…100 If the interactions favor protein folding/stability, reduction of such interactions would destabilize the protein, partially expose the hydrophobic patches, and lead to increased protein aggregation. For example, the rate of thermally induced holo -α -lactalbumin aggregation at pH 7.0 increased with increasing NaCl concentrations from 0 to 60 mM.…”

“…The initial aggregation rate of β -lactoglobulin at pH 5.0 at ambient temperature decreased with increasing ionic strength from 0.0045 to 0.1 M. 165 The aggregation rate of both glycosylated and deglycosylated P. lycii phytase (Phy) at the denaturation temperature was reduced when NaCl was increased from 0 to 0.5 M. 67 More complex relationships have also been reported between protein aggregation and ionic strength. Giger et al 100 studied the aggregation of insulin at pH 5.6 (pI = 5.5) and found that the aggregation (turbidity) of insulin at 0.1 mg/mL at room temperature was fastest in a 10 mM NaCl solution and was reduced at other ionic strengths such as 0.5, 3.0, 30.0, or 100.0 mM. The aggregation of apoferritin at pH 5.0 is bell shaped with increasing Na + concentrations, which could be explained by a well -shaped relationship between B 22 and Na + concentration.…”

Cyclic voltammetry: A new strategy for the evaluation of oxidative damage to bovine insulin

“…The anodic peak arises from the formation of AuO x and the cathodic peak corresponds to the reductive process. 20,21 Compared with curve A, bovine insulin (Fig. 1, curve B) has an increased anodic peak near 1.20 V, but a stable cathodic peak near 0.70 V. So this anodic peak should not only be attributed to the electrochemical oxidation of Au electrode but also be assigned to the oxidation of insulin.…”