| Literature data, reporting the kinetic reaction rate order of the thermal denaturation of β-lactoglobulin (β-LG), were reviewed using two statistical approaches including R2 and the Box-Cox transformation. Although R2, the coefficient of determination, has been used by many researchers as a criterion to compare the fit of data to various kinetic reaction rate orders, this statistic should not be compared for transformations of the same data set. The Box-Cox transformation, however, was constructed to systematically approach the problem of choosing a data transformation, which is the basis for the different reaction rate orders. The results of the analysis showed that, for some data sets, the reaction rate order determined using R2 was not in agreement with the Box-Cox determination. Just as importantly, the Box-Cox transformation resolved that for most data sets analyzed, the reaction rate order was indeterminate and that more than one reaction rate order could represent the data. The conflicting results reported in the literature on the thermal denaturation of β-LG could result from the statistical approach used to analyze the data and determine a reaction rate order.; Subsequently, 3 thermal denaturation studies were conducted using heated solutions of commercial β-LG Fast Protein Liquid Chromatography (FPLC) anion-exchange or gel-permeation chromatography were used to quantify changes in remaining dimeric β-LG. Using the Box-Cox transformation approach, and associated 95% confidence interval surrounding this statistic, it was found that the resulting data could be described by a first-order kinetic reaction rate equation.; Published studies, using size-exclusion chromatography (SEC), have revealed that progressive heating of β-lactoglobulin (β-LG) solutions initially results in the dissociation of β-LG dimers into monomers. In this study, early stages of heat-induced aggregation of β-LG were investigated using FPLC SEC and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). MALDI-MS indirectly confirmed the formation of monomeric protein as measured by FPLC SEC. However, the concentration of monomeric β-LG could be increased or decreased depending on the solution pH, protein concentration, protein source, or mobile phase composition. Under certain conditions, there was no evidence of monomer formation in heated samples of β-LG at or near neutral pH when analyzed using SEC. |
