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      • Study on oligomerization of glutamate decarboxylase from <i>Lactobacillus brevis</i> using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques

        Choi, Jaeyeong,Lee, Seungho,Linares-Pasté,n, Javier A.,Nilsson, Lars Springer Berlin Heidelberg 2018 Analytical and bioanalytical chemistry Vol.410 No.2

        <P>In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (<I>Lb</I>GadB) from <I>Lactobacillus brevis</I> (<I>L</I>. <I>brevis</I>). AF4 provided molecular weight (<I>MW</I>) (or size)-based separation of dimer, hexamer, and aggregates of <I>Lb</I>GadB. The effect of pH on oligomerization of <I>Lb</I>GadB was investigated, and then AF4 results were compared to those from molecular modeling. The <I>MW</I>s measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of <I>Lb</I>GadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of <I>Lb</I>GadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of <I>Lb</I>GadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of <I>Lb</I>GadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1007/s00216-017-0735-6) contains supplementary material, which is available to authorized users.</P>

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