Confocal microscopy study of uptake kinetics of alpha-lactalbumin and beta-lactoglobulin onto the cation-exchanger SP Sepharose FF.
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.
El-Sayed
Mayyada M H
MM
Laboratories of Cambridge Unit for Bioscience Engineering, University of Cambridge, Cambridge, UK.
Chase
Howard A
HA
D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000302:isolation & purification*; D007782:Lactoglobulins / Q000302:isolation & purification*; D018613:Microscopy, Confocal; D008894:Milk Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry*; D013997:Time Factors; D000067816:Whey Proteins
D002412:Cations; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D008894:Milk Proteins; D000067816:Whey Proteins; C097574:sepharose fast flow SP; D012685:Sepharose; D007768:Lactalbumin