In affinity precipitation reversibly water-soluble affinity macroligands, i
.e., polymers bearing an affinity tag, are used to first bind and than co-p
recipitate the target molecule. In this paper a new type of affinity macrol
igand is introduced. The molecules were constructed by group transfer polym
erization of N,N-diethylacrylamide. They were small (less than 15 monomeric
units per molecule) and very homogeneous in size (polydispersity of the mo
lecular mass <1.2). Each base polymer started with carboxylic acid group to
which site the affinity tag (the protease inhibitor m-aminophenylboric aci
d) was later bound by carbodiimid coupling (yield >50%). An inhibition cons
tant, K-1, of less than 10 mu mol/l was determined for the final affinity m
acroligand. Low temperature and high salt concentrations were shown to be b
eneficial to binding. Successful affinity precipitation of the serin protea
se Substilisin carlsberg required a further lowering of the affinity consta
nt by at least one-order of magnitude, which was accomplished by the additi
on of 5% (v/v) of ethylene glycol. The ethylene glycol effect was assumed t
o be due to the formation of a cyclic ester with the phenylboric acid of th
e affinity ligand. (C) 1998 Elsevier Science B.V. All rights reserved.