Tyrosine fluorescence analysis of apolipophorin III-lipopolysaccharide interaction |
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Authors: | Leon Leonardo J Pratt Cindy C Vasquez Lesley J Weers Paul M M |
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Affiliation: | Department of Chemistry and Biochemistry, California State University Long Beach, Long Beach, CA 90840, USA. |
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Abstract: | Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that binds to lipopolysaccharides (LPS). Polyacrylamide gel electrophoresis analysis demonstrated that apoLp-III from Galleria mellonella associated with various truncated LPS variants, including lipid A. Subsequent binding studies were performed employing the intrinsic tyrosine fluorescence properties of apoLp-III, which is highly quenched in the unbound state. A marked increase in tyrosine fluorescence intensity was observed upon binding to LPS or detoxified LPS, indicating a new microenvironment for Tyr-142. This also implies that the LPS carbohydrate region is involved in LPS binding. Dissociation constants (Kd) measured by apoLp-III titration were estimated at approximately 1 microM. Increasing the ionic strength did not decrease the Kd, neither did LPS phosphate removal. In addition, truncation apoLp-III mutants, lacking two complete helices, were still able to associate with LPS. This indicates that the association of apoLp-III with LPS may not be governed by charge but by hydrophobic interactions. |
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Keywords: | Apolipoprotein Apolipophorin Lipopolysaccharides Lipoprotein Tyrosine fluorescence Galleria mellonella |
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