Download Books (18684K PDF)

40 Qjayalakshmi
Table 2
The IgG content and Purity of Different Starting Preparations
Preciprtahon Yield, Purity, Purka Purity %
method % % fold After HLAC
(NH& SO, (O-50%) 88 26 1.88 75
Ethanol Fra. S2 84 58 4.16 95.6
Ethanol Fra. S4 58 90 6.45 98.9
T’unficatlon fold 1s calculated with reference to a chloroform extract Purity of chloro-
form extract, 14%, NaCl extract 1 3%.
Table 3
The Capacity of the Hrsttdyl Sepharose 4B
for Different Protems Testeda
Protein Capacity mg/mL
IgCl 0.5
Myxalme 0.5-l .o
Chymosm 1.0
Tapaclty 1s calculated from the amount of
pure product recovered m real expenments
where crude fractions contammg other pro-
tems/peptldes and molecules were injected
Typically, 7 mL of extract containing 40 mg total protein is
injected on a 35mL column at an average linear flow of 20 cm/h.
7. The specific retention of proteins onto a histidinecoupled adsorbent
(e.g., histidylSepharose or histidyl-silica) is rather closely related to
their isoelectric pH. Thus, only the IgGI, which has a p1 of 8.0 t 0.05,
is selectively retained on a histidyl-Sepharose column at pH 7.4. Be-
low pH 7.4, no IgG fractions are retained (2). Similarly, other pro
teins/peptides that are successfully purified on histidyl-Sepharose
columns are retained only at pH values at or around their isoelectric
pH (Table 4). So, ideally, the equilibrating adsorption pH for a given
protein is chosen, based on the knowledge of their pI values.
8. The theoretical consideration and understanding of the
mechanism(s) of interaction between the proteins/peptides and the
matrix coupled histidine ligands have shown the mechanism to be
water-mediated, involving the changes in the dielectric constants at
the adsorption interface owing to the combined electrostatic, hydra
phobic, and to some extent, charge transfer interactions between his-