N.L.Kalinin

1
N.L.Kalinin

• Serum-ALFA Inc,
• Russian Federation, Moscow. e-mail
  serum2002_at_mail.ru

2
Investigation of protein-carbohydrate
interactions based on thermodynamic constants of
association and dissociation.

• It is well known that protein-carbohydrate
  interaction is fundamental basement for a
  numerous of diagnostic and biotechnological
  methods in clinical medicine and molecular
  biology. Almost of them are considering only
  structural properties (epitop) of carbohydrates
  as part of glycoproteins and glycoconjugates and
  targets for binding..
• In our presentation we would like to suggest
  another possible way to characterize this
  phenomenon. To answer the always main question
  it is specific or nonspecific interaction?
• Our approach is to investigate of thermodynamics
  of the binding . This is discussing in our
  presentation.
• It was shown that each special type of protein-
  carbohydrate interaction may be characterized by
  his own thermodynamic constants of association
  and dissociation.
• Using Biacore technique for studying of
  interactions Mannan binding protein with mannose
  and Con A with glucose the 6 (3 for each pair)
  different constants were calculated. Preliminary
  it may be concluded that these constants are
  markers for recognition step - primary
  interaction, binding step and reception.
• For example Carbohydrates on outer cell membrane
  are carriers of recognition information during
  ontogenesis and cell interaction. Our approach
  may be useful to study in vivo such model
  biological systems as selectin, integrin and
  adhesion processes during cell differentiation
  and proliferation (immune response and tumor
  growth) as well as antigen-antibody binding,
  receptor-ligand and enzyme-substrate
  interactions by in vitro diagnostics.

3
Optiko-biophysical essence of method and typical
experimental curves.
4
Dependence of change of sensogrammy and constants
of association (Kacc) on a temperature.
5
Constants of association (K-ass) and dissociation
(K-diss) of complexes of mannans binding protein
(MBP) and agglyutinin of soy (AS)
6
Suppression of specific relating activity of
immobilization MBP mono- and disaccharide.
7

• FIG. 1. First-order plots of the kinetics of (a)
  association and (b) dissociation for the
  interaction of concanavalin A with the
  carboxy-methyldextran coating on the biosensor
  chip. results from experiments with injected
  lectin concentrations of 1.3 and 0.04 mg/ml,
  respectively. Error bars are included to indicate
  the extent to which the curvilinearity could be a
  consequence of experimental uncertainty.

8

• FIG. 2. First-order plots of the kinetics of (a)
  association and (b) dissociation for the
  interaction of human interleukin-6 with
  biospecific receptor covalently attached to the
  biosensor chip. , - results from experiments
  with 10 and 40 ?? interleukin, respectively. The
  broken line in b denotes the theoretical
  dependence (Eq. 4) based on analysis of a in
  terms of pseudo-first-order kinetics (Eq. f3J).

9
Evaluation of the Effective Total Concentration
of Conca-navalin ? (?d) from the Injected
Concentration (CA) and the Equilibrium BIAcore
Response (Re)
10

• FIG. 3. Thermodynamic characterization of the
  interaction between concanavalin A and
  immobilized carboxymethyldextran on the basis of
  the equilibrium surface plasmon resonance
  responses, (a) Evaluation of the results (Table
  1) by nonlinear regression analysis in terms of
  the rectangular hyperbolic relationship for a
  bivalent solute (Eq. 8 with / 2). (b)
  Corresponding analysis by means of Eq. 12, a
  simplified form of Eq. 8 that applies when (CA
  CA) CA. Dotted lines denote the envelope of
  uncertainty associated with each characterization.