A Simplified Chromatographic Approach to Purify Commercially Available Bovine Submaxillary Mucins (BSM)

Abstract

In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of ∼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.

Structural and Mechanical Properties of Thin Films of Bovine Submaxillary Mucin versus Porcine Gastric Mucin on a Hydrophobic Surface in Aqueous Solutions

“…9-17%) than PGM (0.5-1.5%) of the total mucin masses. As the overall size of mucins are determined mainly by the central, glycosylated region than terminal regions, 4,19 higher density of negatively charged motifs in the former for BSM can be directly related to more sensitive changes of Dh distribution according to pH change, even though  potentials of the two mucins were determined to be comparable at both pH values (Figure 1). …”

“…An important feature in the distribution of Dh after the chromatographic purification of both mucins is that large species ranging from a few hundreds to thousands nm in Dh that were observed from arBSM or arPGM disappeared 19 (Dh distributions for the mucin samples prior to the purification are shown in Figure S3, Supporting Information). Thus, in addition to minimizing non-mucinous protein "impurities" from the samples (see Figure S2), a second effect of the chromatographic purification in this study is to homogenize the size of mucin molecules.…”

“…BSM was purified by means of anion exchange chromatography as described in detail in a previous study. 19 PGM was purified according to the same procedure with BSM. 19 However, due to PGM's higher tendency to form aggregates, additional sterile filtration was performed through 1.2 µm sterile filters (hydrophilic polyethersulfone sterile filters; Pall Corporation, Cornwall, UK) after 5 µm sterile filters, three times each, to clarify the solution.…”

“…Firstly, recent studies have shown that impurities present in commercially available mucins may have a significant impact on their biophysical properties. 16,19,20 Moreover, the nature and amount of these impurities can vary between different batches. Thus, it would be important to keep the amount of impurities as low 4 as possible.…”

“…For this reason, an additional purification step i.e., anion exchange chromatography, was performed on commercially available mucin samples. 19 Secondly, as mucins are polyanionic macromolecules with various acidic moieties, intra-and intermolecular interactions can be controlled by varying ambient pH and it can directly affect the structural and mechanical characteristics of the films. Thus, the structural and mechanical characteristics of the two mucin films were characterized at both neutral and acidic pH, from the film formation stage to exposure to shear stresses.…”