COMPARISON OF PROCEDURES FOR ATTACHING STEROIDAL GLUCOSIDURONIC ACIDS TO BOVINE SERUM-ALBUMIN

Steroidal glucosiduronic acids can be joined to bovine serum albumin under mild conditions by use of either N-hydroxysuccinimide or isobutyl chloroformate. N-Hydroxysuccinimide is the more efficient of the two coupling agents; 30-40 molecules of hapten are incorporated into a molecule of protein when a two-fold excess of glycosiduronate to ε{lunate}-amino groups is used. Conjugates of steroidal glucosiduronic acids and bovine serum albumin which are prepared by use of 1,1'-carbonyldiimidazole tend to lose hapten over a period of days; in addition, use of sodium dodecyl sulfate is required to keep the reactants in solution during preparation of the conjugate. When employed as coupling agents under a variety of conditions, neither of two water-soluble carbodiimides gave a soluble product that consisted of more than 2 molecules of steroidal glucosiduronic acid per molecule of protein. Removal of impurities of low molecular weight from the coupling reactions is more rapid and more efficient when chromatography on Sephadex G-25 is used than when dialysis is employed. Solutions of conjugates should be free of suspended matter if they are to be analyzed by spectroscopic methods. © 1979.