Analysis of Interactions between Carbohydrates and Proteins

 Interactions between carbohydrates and proteins are usually analyzed by labeling protein or carbohydrate (or glycoconjugates) ligands with radioisotope or enzymes. Surface plasmon resonance (SPR), electrospry ionization mass spectrometry (ESI-MS) or nuclear magnetic resonance (NMR) are used without labeling. In recent years, however, time-resolved fluorometry (TRF) using sugar chains labeled with chelated lanthanide is gaining popularity as an easy and sensitive analytical method.

Time-resolved fluorometry

Some lanthanide, especially europium (Eu) and samarium (Sm), can form highly fluorescent chelates, which have a very long fluorescence decay time and large Stokes’ shift. Time-resolved fluorometry is a detection technology based on these unique fluorescence properties of the lanthanide chelates. It allows sensitive and specific detection of lanthanide chelates by eliminating or reducing non-specific fluorescence by pulsing excitation light. Because the lanthanide chelates can be measured as sensitively as radioisotopes and are stable and easy to handle, they are widely used as tracers in the biological sciences. Time-resolved fluoroimmunoassay (TR-FIA) (1) in which Eu-labeled antibodies are used is the most common application.

1. Lanthanide labeling reagents (Fig. 1)
1) Polycarboxylates
DTPA anhydrate (A) (2) and N1(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (B) --- The polycarboxylate-type chelates of Eu normally emit little fluorescence. However, after immuno-reactions, Eu is dissociated from the labeled compound using an enhancement solution (trioctylphosphine oxide, 2-naphthoyltrifluoroacetone, Triton X-100) to form a new and highly fluorescent chelate (See C). This technology is referred to as dissociation enhanced lanthanide fluoroimmunoassay (DELFIA).

2) beta-Diketons
BHHCT (D) (3)------beta-Diketon-type chelates emit high fluorescence, and BHHCT can react directly with proteins.

3) Aryl amines
Quantum-dye (QD) (E)-------Fluorescence of Eu in QD is strong enough for TRF, but it can be further amplified by addition of the enhancement solution.

2. Applications in glycobiology

1) Assay of carbohydrate-related enzymes
There are some reports on glycosyltransferase assays using TR-FIA (4). Activities of glycosyltransferases (5), endoglycosidase and glycopeptidase (6) can also be measured using Eu-labeled lectins.

2) Interactions between carbohydrates and proteins
The use of Eu-labeled glycoproteins and neoglycoproteins in TRF makes it possible to analyze binding specificity of lectins. Lee et al. (5) reported that binding and inhibition assays of carbohydrate-recognition domain of rat serum monnose-binding protein coated on microtiter plates using QD-labeled neoglycoprotein, QD-Man-BSA, yielded results comparable to those obtained using 125I-Man-BSA. TRF is applicable to the assay of hepatic Gal/GalNAc receptor using QD-Gal-BSA. Recently, Oda et al. (7) determined binding specificity of Crocus sativus lectin (CSL) with solid phase method with Eu-labeled (Man)3GlcNAc-glycopepide as well as flow injection, fluorescence polarization and SPR.
Nana Kawasaki ( National Institute of Health Sciences. Division of Biological Chemistry
and Biologicals)
References (1) Hemmlila, I, Dakubu, S, Mukkala, V.-M, Siitari, H, Lovgren, T, Anal. Biochem. 137, 335-343,1984
(2) Kawasaki, N, Lee, YC, Anal. Biochem. 250, 260-262, 1997
(3) Yuan, J, Matsumoto, K, Kimura, H, Anal. Chem. 70, 596-601,1998
(4) Taki, T, Nishiwaki, S, Handa, N, Hattori, N, Handa, S, Anal. Biochem. 219, 104-108 (1994)
(5) Lee, YC, Kawasaki, N, Lee, RT, Suzuki, N, Glycobiology 8, 849-856 (1998)
(6) Deras, I L, Sano, M, Kato, I, Lee, YC, Anal. Biochem. In press.
(7) Oda, Y, Nakayama, K, Kinoshita, M, Kawasaki, N, Hayakawa, T, Kakehi, K, Abdul-Rahman, B, Lee, YC, Glycobiology(abstract), 9, 1132 (1999)
Mar.15, 2000

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