Enzyme Assays on Chips

Introduction Enzyme assays are used for discovery and characterization of enzymes Identification of protein function instead of presence Simultaneous analysis of thousands of samples Small sample volumes required

Array Types Protein array Proteins immobilized Simultaneous examination of enzymatic activity of individual proteins Peptide array Peptide substrates immobilized Search for specific substrate of one enzyme Small molecule array Small molecules (often synthesized) immobilized E.g. fluorogenic substrates of enzymes

Requirements Proper folding and orientation of immobilized enzymes or substrates Solvent (presence of ions, hydrophilicity etc.) Cofactors pH Temperature This makes protein/peptide immobilization more difficult than DNA immobilization

Noncovalent Immobilization Physical adsorption Mainly hydrophobic interactions Nonspecific Capturing methods Biotin-Avidin  site-specific Antibody-antigen  site-specificity depends on method Antibody developed against enzyme Antibody developed against antigen bound to enzyme Polyclonal  several epitopes; monoclonal  one epitope His-tag binding to nickel-nitrilotriacetic acid Entrapment in gels Immobilized molecules in aqueous environment Long incubation time required

Biotin-Streptavidin Binding of biotin to streptavidin. The ureido group of biotin is polarized during binding, whereby the acquired negative charge of oxygen can be stabilized in the oxyanion hole formed by Asn23, Ser27 and Tyr43. Other polar residues in streptavidin form hydrogen bonds to biotin to stabilize the binding further [Weber et al., 1992].

Covalent Immobilization Chemical immobilization EDC/NHS Thiol–thiol (formation of disulphide) or thiol-gold Michael reaction between nucleophile and ,β- unsaturated carbonyl compound Many others exist, including variations of the techniques mentioned above Blocking agents (e.g. BSA) can be used to hinder unspecific binding

EDC/NHS Immobilization In the first step, EDC reacts with the carboxyl group attached to the carrier surface (left). This creates an unstable O-acyl isourea intermediate, and to avoid hydrolysis of the intermediate, NHS is added (middle). Hereby, a stable activated NHS ester is formed and a soluble urea byproduct is released (right) [Vaughan et al., 1999]. When a biomolecule (Bm) is introduced to the activated NHS ester, NHS is replaced by the biomolecule, and immobilization is completed [Vaughan et al., 1999].

Noncovalent vs Covalent Noncovalent Often weaker than covalent methods Usually nonspecific Antibodies can be produced against any antigen Covalent Strong immobilization Site-specific approaches easier  proper orientation

Covalent Biotinylation Intein-mediated site-specific biotinylation Intein at protein C-terminus When the intein is spliced out a thioester is created at the C-terminus, which is then able to react with cysteine-biotin Method A: In vitro in cells (cell lysed before biotinylation) - Chitin-binding domain on intein functions as affinity tag before biotinylation Method B: In vivo in cells (biotinylation inside the cells) Method C: Cell-free system

Detection of Enzymatic Activity Previously enzyme activity could not be detected, since only the binding and not the catalytic activity were detected Detection of enzyme catalytic activity is possible e.g. by formation of colorimetric products Can be used to create substrate “fingerprint” profiles for each enzyme  reveals type of chemical compounds accepted by enzyme, or type of enzyme Using different assay conditions (resembling cytoplasm etc.) reveals complex biological pathways of the enzyme

Detection Methods Colorimetric methods widely used due to simplicity, reliability and sensitivity Fluorescence preferred over radioactive and chemiluminescent methods Radioactivity: Dangerous Chemiluminescence: Limited dynamic range Fluorescence: High signal-to-noise ratio; compatible with standard microarray scanners

Enzyme Inhibitor Identification Inhibitors or specific enzymes can be detected by using fluorescently labeled inhibitors Protein arrays

Enzyme Inhibitor Identification Probes react in time- and concentration-dependent manner  obtain quantitative kinetic data for the enzyme activity Can also be used to detect activators Found inhibitors and activators can be used in the development of novel therapeutics

Detection of Enzyme Activity Proteases, esterases, lipases: Utilize cleavage activity to obtain fluorescent probe Kinases: Fluorescently labeled antibody binds to phosphorylated probe Peptide/small molecule arrays Coumarin