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Separation of the conjugate constructs from unreacted protein, CA and small molecular weight reagents was attempted by GFC. This non-destructive method would allow for the isolation of the constructs without irreversible dénaturation of the protein as seen with TCA precipitation. In theory, if both or at least one of the molecules were in the fractionation range of the separation media, due to differences in their MW, they would be separated by GFC. However, PLi could not be conveniently resolved from CA even though a wide range of separation media (Sephadex G-lOO, Sephadex G-200 and Sepharose CL-6B) was tried. Data are not shown but CA and PLi elution profiles

were systematically overlapped when they were passed separately through the column, probably because the difference in their MW was not sufficient for resolution to occur. Another reason could be the polydisperse nature of both PL and CA which led to broad elution profiles. The use of a higher molecular weight PL would, in theory, solve the problem and PL2 was tried instead. Although the resolution was in fact improved, there was always partial overlapping of the elution profiles, as shown in Fig. 3.5.

o B yxmflmniî

10

20

30

40

50

60

B

^

0.42

0.28

10

20

30

40

50

Elution volume (ml)

60

Fig. 3.5 Molecular sieve chromatography of colominic acid (A) and PL2 (B) at zero time

(O) and after reaction for 11 days (□). Samples were chromatographed on Sepharose CL- 63 (column, 55.0 x 0.9 cm; sample volume, 500 pi; eluent, 0.5 M PBS; flow rate, 20 ml h ’). Arrow indicates the void volume (V o ) of the column. For other details see the Materials and

Comparison of the elution profiles of CA (Fig. 3.5A) at zero time, i.e.

immediately after mixing with PL and at the end of incubation for 11 days at 35-40°C, shows a distinct shift into a higher MW. Elution profiles and retention volumes obtained when CA or PL were chromatographed separately are similar to those obtained at zero time, thus excluding the possibility of ionic interaction between these macromolecules of opposite charge. Another indication of the covalent linkage of the two molecules is that CA assumes the same elution profile as PL2. A smaller proportion

of the conjugates eluted in the void volume (Vo) of the Sepharose CL-6B column, as

indicated by the arrow in Fig. 3.5A. The broad elution peaks obtained are characteristic of polymeric mixtures. In fact, when CA is conjugated to PL, the heterogeneity of the constructs obtained can be due to variations in (a) length of the PL molecule, (b) length of the CA chain attached, (c) number of CA molecules per PL molecule and (d) location of the CA molecules. Even PL molecules with the same degree of modification can exhibit distinct behaviour in GFC, due to differences in shape derived from different points of attachment.

In an independent set of experiments, the coupling reaction was followed over the same period of time by GFC on Sepharose CL-6B. Eluted fractions were assayed for CA and protein contents. Results of elution profiles and elution volumes obtained for CA are shown in Table 3.3 and Fig. 3.6. As before, the elution profile of CA was broader for the sample (A2) where coupling reaction took place. The PL2 elution profile was also broadened in time with its elution peak shifted from fraction 37 to 31 (data not shown). CA and PL2 elution patterns were coincident but the peak of higher MW species that eluted in the void volume of the column in the previous experiment (Fig. 3.5) could not be reproduced. The retention of both elution profile and elution volume of CA in samples B and C, throughout the incubation period, is consistent with the very

low degree of modification of equivalent samples, as determined by TCA precipitation of the conjugates (see Fig. 3.4).

Table 3.2 Range o f colominic acid elution profile (fraction numbers) and elution volumes (ml) in parentheses at different reaction times. Samples contained: A2, CA + PL2 + NaCNBHg (35-

40°C); B, CA + PL2 + NaCNBHg (4°C); C , non-oxidized CA + PL2 + NaCNBHg (35-40°C); D, CA + NaCNBHs (35-40°C). At time intervals, aliquots (500 pi) were centrifuged (SOOOxg for 30 min) and the supernatant applied to a Sepharose CL-6B column (column, 55.0 x 0.9 cm; eluent, 0.5 M sodium phosphate buffer, pH 7.4; flow rate, 20 ml h’’). The void volume (V o ) o f the column (determined with blue dextran) was 20 ml and the elution volume o f unreacted colominic acid in the same calibrated column was 43 ml.

Sample

Reaction time (days)

1 3 5 8 11

A2 22-51 (40) 20-50 (37) 20-51 (37) 21-51 (37) 25-50 (38)

B 32-54 (43) 32-54 (43) 32-54 (43) 32-54 (43) 32-52 (43) C 33-53 (43) 32-54 (43) 32-54 (43) 32-54 (42) 32-54 (42) D 31-55 (43) 32-55 (43) 32-53 (43) 32-53 (43) 34-51 (43)

To rule out the possibility of CA polymerization under the reaction conditions used, which would account for the broadening of its elution profile in this set of experiments, CA was incubated for the same period of time at 35-40°C in the presence of the reducing agent and toluene (preparation D). No modification in the chromatographic behaviour of CA was seen (Table 3.2 and Fig. 3.6) and retention volume (Ve=43 ml) of the polysaccharide after 11 days of incubation, was still coincident with that of oxidized CA passed through the same column. Therefore, CA

CA 0.75 - 0.60 - 0.45 - 0.30 - 0.15 -

Elution volume (ml)

Fig. 3.6 Elution profile o f colominic acid subject to different reaction conditions. Preparations: oxidized CA, PL2 and NaCNBHs (A2) were incubated at 35-40°C (■) or B, 4°C ( □ ) for 11 days; controls containing non-oxidized CA, PL] and NaCNBHs (C) (O ) or CA and NaCNBHs (D) ( • ) were incubated at 35-40°C for the same period. Samples were chromatographed on Sepharose CL-6B (column, 55.0 x 0.9 cm; sample volume, 500 pi; eluent, 0.5 M sodium phosphate buffer, pH 7.4; flow rate, 20 ml h '). The void volume (V o )

o f the column and elution volume o f unreacted oxidized colominic acid (CA) in the same calibrated column are marked with arrows.