3. Results
3.1. Characterization of arabinogalactan by gel filtration chromatography
on Sephacryl S300
3.1.1. Evidence of two components of arabinogalactan
As reported in the Introduction section (1.6.2.), arabinogalactan (AG) used in this study contains different components that can be revealed at different ionic strength. When AG was chromatographed in 10 mM phosphate buffer pH 7 (standard buffer), a single peak was observed (Fig. 3.1, A), while lowering the ionic strength (deionised water), two peaks were revealed (Fig. 3.1, B).
Figure 3.1. Elution profiles on Sephacryl S300 of AG. The elution profiles of gel
filtration chromatography on Sephacryl S300 was performed on a 1,6 cm x 83 cm column at a flow rate of 20 ml/h and fractions of 1,5 ml were collected and analysed for carbohydrate content. Panel A e B refer to the elution profiles of 2 ml of AG (2 mg/ml), performed in standard buffer and deionised water respectively. Peak I (69 ml to 91 ml elution volume) and peak II (108 ml to 139 ml elution volume) were collected, concentrated down to a volume of 2 ml and analyzed separately by using deionised water as eluant. Panel C refers to the superimposition of elution profiles of the two samples: peak I (circles) and peak II (triangles).
The two peaks (I and II), were collected, concentrated on Amicon YM10 membrane and run separately on a column pre-equilibrated with deionised water (Fig. 3.1, C). While peak II sample eluted as a unique peak at the same retention time with respect to what observed in panel B, the peak I sample, mainly recovered at the same retention time with respect to what observed in panel B, displayed also a second even though modest peak (9,7 % of the total absorbance) at a retention time of peak II.
3.1.2. Behaviour of FITC-AG on gel filtration chromatography on Sephacryl S300
When FITC labelled AG (prepared as described in Materials and Methods section) was chromatographed on Sephacryl S300 using standard buffer as eluant, displayed a single peak with the same retention time of the unmodified AG (Fig. 3.2, A). When the chromatography was performed in deionised water, a single peak with lower retention time was observed (Fig. 3.2, B).
Figure 3.2. Elution profiles on Sephacryl S300 of FITC-AG. The elution profiles of gel
filtration chromatography on Sephacryl S300 was performed on 2ml of FIT-AG (2mg/ml) applied on a 1,6 cm x 83 cm column at a flow rate of 20 ml/h either in standard buffer (Panel A) or in deionised water (Panels B); fractions of 1,5 ml were collected and analysed for carbohydrate content.
3.2. Mucin adhesion on different supports and evaluation of the interaction
with arabinogalactan
In order to study the interaction between mucin and AG the protein immobilization on insoluble supports approach was performed. Different conditions and supports suitable for
mucin immobilization are now reported. However no one of them was useful to evaluate the AG:mucin interaction.
3.2.1. Electrostatic interactions on ionic supports
The occurrence of electrostatic interactions between mucin and ionic supports such as DEAE-cellulose, polylysine-coated glass, Dowex 50WX2 and Dowex 88 was evaluated. One ml of slurried DEAE-cellulose (approximately 1,10 g wet weight) pre-equilibrated in standard buffer was added to 4 ml of MUC1 (1,9 mg/ml in standard buffer) and incubated at 25°C under gentle stirring for 2 hours. After incubation the suspension was centrifuged 1 min at 2000xg and the resin was washed for 4 times by 4 ml of standard buffer. All the supernatants were pooled and the protein content was measured. Only 1 mg on a total of 7,6 mg of MUC1 was detected on supernatants pool, indicating a mucin adhesion on the ionic support of 87 %. Unfortunately the observed high aspecific adhesion of AG to such an anionic exchanger, possibly coming from the polysaccharidic nature of the resin support, combined with the even though modest negative charge density on AG, compromised the study of the interaction between MUC1 and AG. This is clearly evident in Fig. 3.3 in which the interaction of AG with DEAE bound MUC1 was compared with the binding on the free DEAE resin (histograms a and b). Possible contribution of electrostatic interaction between AG and the ionic exchanger comes from the decrease in binding (histogram c) observed with a DEAE-bound to MUC1 previously saturated by a treatment with thyroglobulin (incubation with 11 mg tyroglobulin/ml of resin for 2 h plus 4 washing steps).
Figure 3.3. Interaction between AG and MUC1 immobilized on DEAE-cellulose. The pellet of 1 ml of slurried
resin was suspended in 2 ml of 0,2 mg/ml of AG in standard buffer and incubated at 25°C under gentle stirring. After 1 h, the suspension was centrifuged and AG measured on the supernatant. Such a value was subtracted from total AG in order to evaluate the AG bound to the resin. Histograms refer to: a) free cellulose, b) DEAE-cellulose bound to MUC1 and c) DEAE-DEAE-cellulose bound to MUC1 saturated with thyroglobulin. Error bars
Another anionic exchanger characterized by a less hydrophilic matrix, as polylysine coated glass, was tested. Polylysine coated glass (2,6 x 7,6 x 0,1 cm) were placed in 3,75 ml of 0,3 mg/ml MUC1 in standard buffer and incubated at 37°C under gentle stirring for 6 days. At different times glasses were rinsed in standard buffer and the protein content was evaluated by bicinchoninic acid method (see Methods section) (Fig. 3.4, A). The interaction between immobilized mucin and AG was tested by FITC-AG (Fig. 3.4, B). Also in this case, an aspecific high AG adhesion was observed, which compromised the evaluation of the MUC1-AG interaction (Fig. 3.4, B).
In order to counteract the possible contribution of charge interaction between AG and the support, attempts were done in order to bound mucin to a cationic exchanger. However no conditions were found to attach MUC1 to Dowex 50WX2 and Dowex 88 in the pH range from 3 to 8.
Figure 3.4. MUC1 adhesion on polylysine-coated glass and interaction with AG. Panel A: time course of MUC1
adhesion on polylysine-coated glass (see text for details). Panel B: FITC-AG binding on free polylysine-coated glass (a) and on the MUC1 immobilized polylysine-coated glass (b). The assay was performed by incubating the support layers at 25°C in 3 ml of 0,1 µg/ml FITC-AG for 3 h before to measure the solution fluorescence. Bound AG was evaluated by subtracting the fluorescence after 3 h of incubation to the initial fluorescence value. Error bars represent SEM.
3.2.2. Non-ionic interaction
To minimize the aspecific adhesion of AG on supports, non ionic supports were investigated. Several polystyrene and polyvinyl chloride (PVC) commercial supports, such as plates, micro wells, beads, thick and thin layers were tested and the most promising adopted were polystyrene plates and PVC thick layers. A volume of 3,75 ml of 0,3 mg/ml MUC1 in standard buffer was incubated at 37°C under gentle stirring for 6 days on either polystyrene plates (Ø 4 cm) or on PVC thick layers (2,5 x 7,5 x 0,05 cm). At different times, the supports were rinsed in standard buffer and the protein-bound content was evaluated by bicinchoninic acid method
(see Methods section) (Fig. 3.5, A). The maximal level of mucin adhesion on polystyrene was 0,10 µg/cm2 of MUC1 after 3 days of incubation. At the same time, the adhesion of MUC1 on PVC layers, not yet maximal, was 0,45 µg/cm2. When assayed for AG binding ability, MUC1 immobilized on PVC layers revealed not to be able to allow a confident evaluation of the MUC1-AG interaction. Also in this case, it was observed a rather high aspecific binding of AG to the free support, so that the observed differences were not statistically significant (Fig. 3.5 B, a and b). On the contrary, the AG binding ability on free polystyrene plates was lower than on free PVC layers. However, since the low amount of MUC1 bound to this support, the quantification of AG adhered on MUC1 immobilized on polystyrene was affected by an high variability (Fig. 3.5 B, c and d).
Figure 3.5. MUC1 adhesion on non ionic supports. Panel A: time course of MUC1 adhesion on polystyrene
plates (circles) and PVC thick layers (squares) (see text for details). Panel B: FITC-AG binding on free PVC layers (a), on the MUC1 immobilized PVC layers (b), on free polystyrene plates (c) and on the MUC1 immobilized polystyrene plates (d). The assay was performed by incubating the supports at 25°C in 3 ml of 0,1
µg/ml FITC-AG for 3 h before to measure the solution fluorescence. Bound AG was evaluate by subtracting the fluorescence after 3 h of incubation to the initial fluorescence value. Error bars represent SEM.
3.2.3. Covalent immobilization
Mucin covalent immobilization on glass fiber filters was performed by incubating glass fiber filters, previously modified as described in “Materials and Methods”, for 16 hours at 37°C in 2,4 ml of 1,5 mg/ml MUC1 solution in standard buffer, containing 1M NaBH4 and 1M NaOH,
(see scheme Fig. 3.6). After the incubation, the glass fiber filters were rinsed in standard buffer and protein content was evaluated by bicinchoninic acid method. This methodological approach revealed to be the most promising to immobilize mucin on a support, allowing in the adopted conditions to bind up to 36 µg/cm2 of MUC1.
Figure 3.6. MUC1 immobilization on glass-fiber filters.
The release of proteic materials from glass filters was tested and as it results from Fig. 3.7 approximately 70% of mucin remains bound to the filter after 6 h of incubation. However, also in this case, the study of the interaction between immobilized-MUC1 and AG was impaired by the high background of AG adhesion on the unactivated support and on activated filters, either free or linked to MUC1 (Fig. 3.8). This is also true when the activated filters, either free or linked to MUC1, were saturated with different amino groups exposing molecules, such as ethanolamine, glycine and TRIS, able to block possibly residual reactive epoxide groups.
Figure 3.7. Release of MUC1 immobilized on glass fiber filters. MUC1 immobilized on glass-fiber filters was
incubated at 25°C. At the indicated times, mucin content immobilized on filters was evaluated by bicinchoninic acid method.
Figure 3.8. Interaction between AG and MUC1 immobilized on glass fiber filters. The assay was performed by
incubating the glass fiber filters at 25°C in 3 ml of 1:50 (w:w) mixture FITC-AG:AG (60 µg/ml) for 3 h before to measure the solution fluorescence. Bound AG was evaluate by subtracting the fluorescence after 3 h of incubation, to the initial fluorescence value. Histograms a, b and c refer to the assay performed on unactivated filters, free activated filters and MUC1-bound filters, respectively. Error bars represent SEM.
3.2.4 Physical entrapment on polyacrilamide gel
Another support tested was polyacrilamide gel, by physical entrapment, but the presence of polyacrilamide interfered with mucin detection analysis, compromising the use of this kind of support.
3.3. Analysis of the interaction between MUC1 and arabinogalactan in
solution
3.3.1 Gel filtration chromatography of MUC1 and arabinogalactan on Sephacryl S300
When MUC1 and AG were chromatographed separately on a Sephacryl S300 column (1,6 x 83 cm), they displayed well distinct elution peaks (Fig. 3.9, A); when the two molecular species were chromatographed together, in the same conditions, a marked change in the elution profiles of both AG and MUC1 was observed (Fig. 3.9, B). In particular, a significant shift of the elution peak of AG toward higher molecular weight and a considerable “tailing” of the mucin peak were observed.
Figure 3.9. Gel filtration chromatography of MUC1 and
arabinogalactan. The elution
profiles of MUC1 and AG on Sephacryl S300 (1,6 cm x 83 cm) are reported. The columns were loaded with 2 ml of sample and eluted with standard buffer at a flow rate of 20 ml/h; fractions of 2 ml were collected. The mucin content was evaluated measuring the absorbance at 280 nm (circles) and the AG content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). Panel A: superimposition of the elution profiles of AG (2 mg/ml) and MUC1 (1 mg/ml); Panel B: elution profile of a mixture of AG (2 mg/ml) and MUC1 (1 mg/ml). In each fraction the contribution of mucin to the absorbance at 620 nm was subtracted.
When an identical chromatographic support is used, while reducing 2 fold the length of the column, a partial superimposition of the elution profiles of MUC1 and AG is observed (Fig. 3.10, A). However, when the two species were chromatographed together, changes in the elution profiles were again observed, as described for Fig. 3.9, B. Both the ratio AG:MUC1 and their absolute concentrations affect the described shift of the elution peak of AG toward higher molecular weight and the “tailing” of the mucin peak. In fact, this overlapping became progressively less evident (Fig 3.10, B and C) or completely absent (Fig. 3.11) when the ratio AG:MUC1 or their concentration were lowered.
Figure 3.10. Gel filtration chromatography of mixtures of AG and MUC1 at different w:w ratios.
The elution profiles of gel filtration chromathography on Sephacryl S300 (1,6 cm x 42 cm) of mixtures of AG and MUC1 at different w:w ratios are reported. The columns were eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The mucin content was evaluated measuring the absorbance at 280 nm (circles) and the AG content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). The green and red profiles refer to elution profiles of MUC1 and AG, respectively, obtained when they were chromathographed separately in the same conditions described in the corresponding panel. The following 2 ml samples were applied to the columns: Panel A: 2 mg MUC1 and 4 mg AG; Panel B: 2 mg MUC1 and 2 mg of AG; Panel C: 2 mg MUC1 and 1 mg AG.
Figure 3.11. Gel filtration chromatography of mixtures of AG and MUC1 at low concentration. The elution
profile of a mixture of AG and MUC1 on Sephacryl S300 (1,6 cm x 83 cm) is reported. The column was loaded with 2 ml of sample, containing a mixture of AG (0,57 mg/ml) and MUC1 (0,29 mg/ml), eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The mucin content was evaluated measuring the absorbance at 280 nm (circles) and the AG content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). The red and green profiles are relative to AG and MUC1, respectively, chromathographed alone in the same conditions described above.
3.3.2. Effect of mucin on the resolution capacity of the gel filtration chromatographic support
The possible effect of mucin on the resolution capacity of the Sephacryl S300 support was tested by comparing the elution profiles of both cytochrome C and chymotrypsinogen in the absence and in the presence of MUC1. As observed in Fig. 3.12, the elution peaks of both cytochrome C and chymotrypsinogen were completely unaffected by the presence of MUC1.
Figure 3.12. Effect of mucin on the resolution capacity of the Sephacryl S300 support. The Sephacryl S300
columns (1,6 cm x 83 cm) were loaded with 2 ml of different mixtures, eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The mucin content was evaluated measuring the absorbance at 280 nm (circles). Panel A: elution profile of a mixture of cytochrome C (2 mg/ml) and MUC1 (1 mg/ml); the cytochrome C content was evaluated measuring the absorbance at 550 nm (triangles). Panel B: elution profile of a mixture of chymotrypsinogen (2 mg/ml) and MUC1 (1 mg/ml); the chymotrypsinogen content was evaluated measuring the absorbance at 280 nm (squares). The red profiles are relative to cytochrome C (panel A) and chymotrypsinogen (panel B) chromathographed alone.
3.3.3. Gel filtration chromatography of arabinogalactan in the presence of high molecular weight proteins
Mixtures containing AG and different proteins (either glycosylated or not) with molecular weights comparable to mucin, such as alpha-crystallin, ferritin and thyroglobulin, were analysed by gel filtration chromatography. As reported in Fig. 3.13, in each case the elution profile of the mixture is superimposable to that obtained when the two molecular species were chromatographed separately.
Figure 3.13. Gel filtration chromatography of AG in the presence of high molecular weight proteins. The
elution profiles of gel filtration chromathography of mixtures of AG and high molecular weigth proteins are reported. The Sephacryl S300 columns (1,6 cm x 83 cm) were loaded with 2 ml of sample, eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The protein content was evaluated measuring the absorbance at 280 nm (squares) and the AG content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). Panel A: mixture of AG (2 mg/ml) and alpha crystallin (1 mg/ml); panel B: mixture of AG (2 mg/ml) and ferritin (1 mg/ml); panel C: mixture of AG (2 mg/ml) and thyroglobulin (1 mg/ml). The red profiles are relative to AG chromathographed alone. The blue profiles, from panel A to C, are relative to alpha crystallin, ferritin and thyroglobulin, chromatographed alone.
3.3.4. Equilibrium dialysis
Equilibrium dialysis using classical cellulose tubing resulted in a high undesirable interaction of AG with tubing (42% of the total AG loaded), which affected the permeation of AG through the membrane. However, neither the use of hydrophobic polyvinylidene fluoride tubing (PVDF) resulted useful for the equilibrium dialysis approach. In fact, as reported in Table 3.1, AG seemed to be strongly absorbed by the dialysis membrane. The presence of NaCl did not ameliorate the AG recovery. This observation is supported by results shown in Fig. 3.14.
sample Solution “IN” Solution “OUT” AG (mg/ml) “OUT” AG (mg/ml) “IN” 1 S-buffer AG 0,495 0,400 (81%) 2 AG AG 0,458 0,324 (71%)
3 S-buffer AG, NaCl 0,471 0,310 (66%)
4 AG, NaCl AG, NaCl 0,498 0,234 (47%)
Table 3.1. Permeation of AG through PVDF dialysis tubing. A PVDF dialysis tubing (0,5 ml) containing either
Standard buffer (S-buffer) (sample 1) or 0,5 mg/ml AG (sample 2) was kept at 4°C for 48 h under gentle stirring in 40 ml of 0,5 mg/ml AG. Sample 3 and 4 refer to the same experimental approach as for sample 1 and 2, respectively, with the addition of 0,15 M NaCl to the AG solution. At the end of the dialysis, AG content, inside (IN) and outside (OUT) the tubing, was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent. Values in parenthesis refer to the percentage of the AG concentration inside with respect to the outside concentration.
Since the non-equilibrium attainment between inside and outside the tubing, the possible adhesion of AG to the membrane was evaluated. AG adhered on PVDF membrane, as shown in Fig. 3.14.
Figure 3.14. AG adhesion on PVDF membranes. PVDF membranes (13 cm2) were incubated at 4°C with 2 ml of
AG (0,5 mg/ml) in standard buffer in the absence (squares) and in the presence of NaCl (0,15 M) (triangles). At different days of incubation the AG content in solution was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent and followed by subtraction from the initial concentration. The results shown the percentage of AG bound.
3.3.5. Frontal gel filtration chromatography on Sephacryl S300
Frontal gel filtration (Winzor et al., 1967) was performed by chromatographing MUC1 on a Sephacryl S300 column using different concentrations of AG, ranging from 0,16 to 0,23 mg/ml, as equilibrating buffer and as eluant. Fig. 3.15 reports the elution profile obtained when MUC1 was subjected to frontal gel filtration chromatography using 0,16 mg/ml AG in the mobile phase. Fig. 3.16 reports, for all the AG concentrations used (AG free in Fig. 3.17), the profile of AG content in fractions corresponding to the MUC1 elution peak. For each concentration of AG, all the fractions whose carbohydrate content was higher with respect to the AG baseline were considered for the evaluation of the concentration of the complex AG-MUC1 (AG bound in Fig. 3.17). The molar concentration of AG was estimated on the basis of a molecular weight for AG of 37 KDa (Ponder and Richards, 1997a). The data, analysed by Schatchard plot, allowed the evaluation of a dissociation constant for the complex AG-MUC1 of 5,20 ± 0,55 x 10-6M (Fig. 3.17).
Figure 3.15. Frontal gel filtration chromatography of MUC1 using AG as eluant. An aliquot (2 ml) of MUC1 (1
mg/ml) was applied to a column of Sephacryl S300 (1,6 cm x 83 cm); 0,16 mg/ml of AG in standard buffer was used as equilibrating buffer and eluant. The flow rate was of 20 ml/h and fractions of 2 ml were collected. Mucin content was estimated by measuring the absorbance at 280 nm; AG content was estimate by measuring the absorbance at 620 nm after reaction with the anthrone reagent. In each fraction the contribution of mucin to the absorbance at 620 nm was subtracted.
Figure 3.16. AG evaluation in fractions from frontal gel filtration chromatography of MUC1. Frontal gel
filtration of MUC1 was performed on Sehacryl S300 as described in Fig. 3.15 using as eluant the indicated concentrations of AG. The reference position of MUC1 in the eluted fractions is indicated.
Figure 3.17. Determination of the dissociation constant of the AG-MUC1 complex by Schatchard plot. Each
point is obtained from a single frontal gel filtration chromatography. See text for details. By linear regression, the binding constant was estimated.
3.3.6. Interaction of AG-I and AG-II with MUC1
The AG-I and AG-II fractions were isolated from gel filtration on Sephacryl S300 as shown in Fig. 3.1, B. The behaviour of both components on Sephacryl S300 was analyzed either in the absence or in the presence of MUC1. Results reported in Fig. 3.18 shown that, as observed for AG, both AG-I and AG-II peaks in the presence of MUC1 exhibited a shift toward higher molecular weight. At the same time, a tailing of MUC1 occurred when the protein was chromatographed together with either AG-I or AG-II.
Figure 3.18. Gel filtration
chromatography of AG-I and AG-II in the presence of MUC1. A
Sephacryl S300 column (1,6 cm x 83 cm) was loaded with 2 ml of sample, eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The protein content was evaluated measuring the absorbance at 280 nm (circles) and the AG content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). Panel A: elution profile of a mixture of 1 mg/ml of MUC1 and 1 mg/ml of AG-I. Panel B: elution profile of a mixture of 1 mg/ml of MUC1 and 1 mg/ml of AG-II. Red profiles in Panel A and B refer to elution profiles of AG-I and AG-II (2 mg in 2 ml of sample), respectively, chromatographed alone. Green profiles refer to elution profile of MUC1 (2 mg in 2 ml) chromatographed alone.
3.4. Analysis of the interaction of AG with MUC2 and MUC3
When MUC2 and MUC3 were analysed by gel filtration chromatography on Sephacryl S300, they both revealed, with respect to MUC1, a more composite elution profile, with the presence of carbohydrate portion associated only to the higher molecular weight fractions (Fig. 3.19).
Figure 3.19. Comparison of elution profiles of different mucins through gel filtration. The elution profiles of
gel filtration chromatography on Sephacryl S300 (1,6 cm x 83 cm) of MUC1 (Panel A), MUC2 (Panel B) and MUC3 (Panel C) are reported. The columns were loaded with 2 ml of 1 mg/ml protein samples, eluted with standard buffer at a flow rate of 20 ml/h. Fractions of 1,5 ml were collected. The carbohydrate content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (circles). Mucin content was evaluated measuring the absorbance at 280 nm (triangles). The red boxes identify the fractions that were collected to obtain high molecular weight pools of the different mucins.
The attempt to evaluate differences in the cross-reactivity toward anti-MUC2 and anti-MUC3 antibodies between glycosylated and not-glycosylated fractions of different mucins, through ELISA test resulted unsuccessful.
The glycosylated high molecular weight fractions of MUC2 and MUC3 (MUC2-G and
A
C B
order of obtain sufficient amounts of both MUC2-G and MUC3-G to analyze their behaviour on gel filtration in the presence of AG. Fig. 3.20 reports the elution profile of a mixture of MUC3-G and AG. It is evident a decrease of the retention time of AG in the presence of MUC3-G; on the other hand, unlike what observed for MUC1, no “tailing” of the MUC3-G peak was observed in the presence of AG (Fig. 3.20, A). Since the high carbohydrate content of both MUC2-G and MUC3-G, the use of FITC-AG resulted fundamental for the specific evaluation of the polysaccharide when it was subjected to gel filtration in the presence of either MUC2-G or MUC3-G. This is particularly evident in Fig. 3.20, B. As observed in the study of the interaction between AG and MUC1 (Paragraph 3.3.1), when the concentration of AG and MUC3-G was lowered, even if maintaining constant their ratio, no changes in the elution profile of the two species were observed when they were chromatographed together (Fig. 3.21). When a mixture of MUC2-G and AG was subjected to gel filtration chromatography, the elution profile of AG appeared almost superimposable to that observed for the polysaccharide chromatographed alone, the only difference being a slight increase in the peak width (Fig. 3.22).
Fig. 3.20. Gel filtration chromatography of a mixture of MUC3-G and AG. A 2 ml
sample containing 2 mg of MUC3-G and 4 mg of a 1:50 (w:w) mixture FITC-AG: AG was applied on a Sephacryl S300 column (1,6 cm x 83 cm). The elution was performed with standard buffer at a flow rate of 20 ml/h and fractions of 1,5 ml were collected. Panel A: mucin content was evaluated by measuring the absorbance at 280 nm (circles) and AG content was evaluated by measuring the emission of fluorescence at 514 nm with an excitation wavelength of 494 nm (squares). Green and red profiles refer to the elution profiles of MUC3-G and AG, respectively, chromatographed alone in the same conditions. Panel B: the carbohydrate content of each fraction was evaluated by measuring the absorbance at 620 nm after reaction with the anthrone reagen (triangles). Blue profile refers to the elution profile of AG chromatographed alone in the same conditions.
Figure 3.21. Gel filtration chromatography of a mixture of MUC3-G and AG at low concentrations. The
elution profiles of gel filtration chromathography on Sephacryl S300 (1,6 cm x 83 cm) are reported. A 2 ml of sample, containing a mixture of AG (0,57 mg/ml) and MUC3-G (0,29 mg/ml) was applied to the column, eluted with standard buffer at a flow rate of 20 ml/h and fractions of 2 ml were collected. The protein content was evaluated measuring the absorbance at 280 nm (circles) and the carbohydrate content was evaluated measuring the absorbance at 620 nm after reaction with the anthrone reagent (triangles). The red and green profiles are relative to AG and MUC3-G, respectively, chromathographed alone in the same conditions.
Figure 3.22. Gel filtration chromatography of a mixture of MUC2-G and AG. A 2 ml sample containing 2 mg of
MUC2-G and 4 mg of a 1:50 (w:w) mixture FITC-AG: AG was applied on a Sephacryl S300 column (1,6 cm x 83 cm). The elution was performed with standard buffer at a flow rate of 20 ml/h and fractions of 1,5 ml were collected. AG content was determined by measuring the emission of fluorescence at 514 nm with an excitation wavelength of 494 nm (triangles) and mucin content was determined by measuring the absorbance at 280 nm (circles). Red profile refers to elution profile of AG chromatographed alone in the same conditions.
