Confocal microscopy study of uptake kinetics of α‐lactalbumin and β‐lactoglobulin onto the cation‐exchanger SP Sepharose FF

Abstract

Confocal laser scanning microscopy (CLSM) was used to study single- and two-component protein uptake for alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG), as models for whey proteins, to SP Sepharose FF at pH 3.7 during batch experiments in a finite bath. By coupling a fluorescent dye with the protein molecule, the penetration into individual adsorbent particles at different times during batch uptake was visualised. In a single-component system, BLG penetrated fast into the adsorbent beads and gradually filled them in a shell-wise fashion, while adsorption of ALA was mostly confined to the outer shells of the adsorbent. For the two-component studies, the results showed that ALA was able to displace BLG despite its lower affinity to the adsorbent under the employed conditions. CLSM results were then compared both qualitatively and quantitatively to their counterparts obtained in traditional experiments by indirect measurements of the protein concentration in the fluid phase. A novel quantitative approach was undertaken by modifying the simple kinetic rate model traditionally used to determine the kinetic rate constant, k(1), for batch uptake experiments, in order to describe batch uptake kinetics based on CLSM data. Although BLG results were in good agreement, there was a discrepancy in ALA results.

Purification of the two major proteins from whey concentrate using a cation‐exchange selective adsorption process

“…The confocal results also showed evidence that the weaker-affinity protein was able to displace BLG. A mechanism compatible with this result was proposed (El-Sayed and Chase, 2009b).…”

Simulation of the breakthrough curves for the adsorption of α-lactalbumin and β-lactoglobulin to SP Sepharose FF cation-exchanger

“…3. The theoretical profile was predicted using the relevant equilibrium isotherm parameters (q m = 147.0 mg/ml and K d = 0.029 mg/ml) [11], and the kinetic rate constant for adsorption within the medium-size particles (k 1 = 30 × 10 −3 ml mg −1 min −1 ) [12]. As can be seen in Fig.…”

“…The value of the rate constant for the smallest particle size was determined using the method outlined in Ref. [12] and was found to be 55 × 10 −3 ml mg −1 min −1 . Hence, the early stage of the breakthrough curve is better predicted using the value of the rate constant corresponding to adsorption within the smallest particles, while the later stage of the curve could be predicted using the value of the rate constant corresponding to adsorption within the medium-size particles (80-90 m).…”

“…The theoretical breakthrough curve (solid curve, Fig. 1A) was obtained using values of the equilibrium isotherm parameters, q m (113.6 mg/ml) and K d (0.008 mg/ml), determined from batch experiments [11], as well as the forward kinetic rate constant for adsorption within medium-size adsorbent particles, k 1 (35 × 10 −3 ml mg −1 min −1 ), obtained from batch uptake experiments [12]. As can be seen from the figure, the experimental adsorption profile (discrete points) in its early stage (before reaching t 1/2 ) yields a sharp and symmetrical breakthrough curve that is steeper than its corresponding theoretical curve.…”

“…In a preceding series of publications [11][12][13][14], we have managed to develop a method for isolating and separating the two major proteins, ␣-lactalbumin and ␤-lactoglobulin, from whey. The method is chromatographic and is based on selective adsorption of the two proteins in cationic forms to the cation-exchanger SP Sepharose FF.…”