Gels Mimicking Antibodies in Their Selective Recognition of Proteins and Its Potential Use for Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 22

Fig. 6. Preparation of a selective bed with higher flow rate. A polyacrylamide column
of the composition T6 C5 and specific for hemoglobin was prepared essentially as
described for the “ribonuclease column” (Fig. 1). The column was emptied and the gran-
ules were suspended in a monomer solution of the composition T20, C3. Following poly-
merization the gel was granulated by pressing it through a 100-mesh net and packed in a
Pasteur pipette. A blank column was prepared in the same way but in the absence of hemo-
globin. A standard solution consisting of hemoglobin (Hb), cytochrome c (C) and
lysozyme (L) was (a) passed through the blank column and analyzed in a cation exchanger,
and (b) passed through the column prepared in the presence of hemoglobin and analyzed
in the ion exchanger. The chromatogram traces indicate that the T6, C5/T20, C3 column
adsorbed hemoglobin. Accordingly, entrapment of the T6, C5 granules into small, rigid
T20, C5 gel grains did not affect the selective recognition of hemoglobin. Owing to the
rigidity of these grains the column had the advantage of permitting high flow rates.
Do some protein molecules become attached
covalently to the gel matrix?
This could be so because “hemoglobin columns” are somewhat colored
even after extensive washing with different buffers and a 10% solution of
acetic acid containing 10% SDS. Since monomer radicals form in the
polymerization process, one cannot exclude the possibility that these react
with hemoglobin molecules, which thus may become linked to the gel
network. To investigate whether such a reaction occurs, a “hemoglobin
column” was prepared as described by Liao et al. (1996) with the excep-
tion that many amino acids were present at high concentrations along with