In Vitro Preparation of Nonenzymatically Glucosylated Human Transferrin, α₂-Macroglobulin, and Fibrinogen with Preservation of Function

Human transferrin, α₂-macroglobulin, and fibrinogen were incubated with [³H]-glucose. After a 7-day, 37°C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; α₂-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 mol of glucose/ mol, or approximately 14 μmol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a K_d = 33 nM and receptor number of 3.4 × 10⁵ receptors/cell; nonglucosylated transferrin with a K_d = 31 nM and receptor number of 3.9 × 10⁵ receptors/cell. Glucosylated and nonglucosylated α₂-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of ¹²⁵I-labeled, methylamine-treatedα₂-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated α₂-macroglobulin, t_1/2 = 3 min. α₂-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a K_d of 2.5 nM and receptor activity of 370 fmol/mg cell protein. α₂-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a K_d of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen. The crosslinking by factor XIII and degradation by plasmin were the same for glucosylated and control fibrin as determined on SDS-PAGE. Glucosylated fibrinogen was degraded by plasmin to yield identical fragments on SDSPAGE as the control fibrinogen; however, glucosylated fibrinogen was degraded at a slightly slower rate. Platelet binding studies showed a K_d of 6.6 × 10⁻⁷ M and receptor number of 33,000 sites per cell for control fibrinogen and a K_d of 6.8 × 10⁻⁷ M and receptor number of 28,000 sites per cell for glucosylated fibrinogen.