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Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins.

ABSTRACT: The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carotenoid preparations that are nano-dispersed in aqueous buffers, and then we used a new-generation surface plasmon resonance (SPR) protocol called FastStep™, which is significantly faster than conventional SPR assays. We have explored carotenoid-binding interactions of five proteins: human serum albumin (HSA), ?-lactoglobulin (LG), steroidogenic acute regulatory domain proteins (StARD1, StARD3) and glutathione S- transferase Pi isoform (GSTP1). HSA and LG showed relatively weak interaction with carotenoids (K(D)>1 ?M). GSTP1 evidenced high affinity and specificity towards zeaxanthin and meso-zeaxanthin with K(D) values 0.14±0.02 ?M and 0.17±0.02 ?M, respectively. StARD3 expressed a relative high specificity towards lutein with a K(D) value of 0.59±0.03 ?M, whereas StARD1 exhibited a relatively low selectivity and affinity (K(D)>1 ?M) towards the various carotenoids tested.

SUBMITTER: Vachali P

PROVIDER: S-EPMC3289994 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins.

Vachali Preejith P Li Binxing B Nelson Kelly K Bernstein Paul S PS

Archives of biochemistry and biophysics 20120120 1

The xanthophyll carotenoids lutein and zeaxanthin constitute the major carotenoids of the macular pigment in the human retina where they are thought to act in part to prevent light induced oxidative damage associated with age-related macular degeneration (AMD). The highly selective uptake of these pigments is mediated by specific carotenoid-binding proteins (GSTP1 and StARD3) recently identified in our laboratory. Carotenoids are hydrophobic in nature, so we first systematically optimized carote ...[more]

PMID: 22286029

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Project description:Lectins are carbohydrate binding proteins found in plants, animals, and microorganisms. They serve as important models for understanding protein-carbohydrate interactions at the molecular level. We report here the fabrication of a novel sensing interface of biotinylated sialosides to probe lectin-carbohydrate interactions using surface plasmon resonance spectroscopy (SPR). The attachment of carbohydrates to the surface using biotin-NeutrAvidin interactions and the implementation of an inert hydrophilic hexaethylene glycol spacer (HEG) between the biotin and the carbohydrate result in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The specificity and sensitivity of lectin binding were characterized using Sambucus nigra agglutinin (SNA) and other lectins including Maackia amurensis lectin (MAL), concanavalin A (Con A), and wheat germ agglutinin (WGA). The results indicate that alpha2,6-linked sialosides exhibit high binding affinity to SNA, while alteration in sialyl linkage and terminal sialic acid structure compromises the affinity by a varied degree. Quantitative analysis yields an equilibrium dissociation constant (KD) of 777 +/- 93 nM for SNA binding to Neu5Ac alpha2,6-LHEB. Transient SPR kinetics confirms the K D value from the equilibrium binding studies. A linear relationship was obtained in the 10-100 microg/mL range with limit of detection of approximately 50 nM. Weak interactions with MAL, Con A, and WGA were also quantified. The control experiment with bovine serum albumin indicates that nonspecific interaction on this surface is insignificant over the concentration range studied. Multiple experiments can be performed on the same substrate using a glycine stripping buffer, which selectively regenerates the surface without damaging the sialoside or the biotin-NeutrAvidin interface. This surface design retains a high degree of native affinity for the carbohydrate motifs, allowing distinction of sialyl linkages and investigation pertaining to the effect of functional group on binding efficiency. It could be easily modified to identify and quantify binding patterns of any low-affinity biologically relevant systems, opening new avenues for probing carbohydrate-protein interactions in real time.

Dataset Information

Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins.

Publications

Surface plasmon resonance (SPR) studies on the interactions of carotenoids and their binding proteins.

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