Hans Sigrist

A hydrophobic form of the small-intestinal sucrase-isomaltase complex

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.

Surface immobilization of biomolecules by light

Biomolecules performing specific biological functions on material surfaces are progressively employed in the development of miniaturized bioassays, biosensors, bioelectronic devices, and medical equipment. Device performance is improved with covalently immobilized bioconstituents. The unique advantages of using light-controlled reactions to achieve biomolecule immobilization on surfaces are addressed. On activation of introduced light-sensitive reagents, biomolecules are covalently linked to material surfaces. Procedures leading to light-dependent engineering of surfaces are exceptionally facile. Immobilization by light is compatible with biological functions, enabling surface patterning and molecular coating of materials. Current strategies and protocols are illustrated with selected examples of biomolecule photoimmobilization.

Photochemical linkage of antibodies to silicon chips.

Antibodies and antigen binding fragments thereof were photochemically immobilized on surface‐modified silicon chips of 5 × 5 mm size. Silicon surface‐grafted diazirines and benzophenones formed covalent bonds with the immunoreagents on light activation. Photolithographic immobilization of monoclonal antibodies in aqueous media was achieved on silicon chips by activating surface‐grafted benzophenones. The presence of bovine serum albumin during irradiation reduced nonspecific adsorption of the immunoreagents and retained the immunoactivity of the photoimmobilized molecules.

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. II. Topology of phenylisothiocyanate binding sites and influence of p-sulfophenylisothiocyanate on phenylisothiocyanate modification

The two structurally related probes, the apolar phenylisothiocyanate and the polar, water-soluble p-sulfophenylisothiocyanate, were analysed for their topological interaction with human erythrocyte band 3 protein. Upon thermolytic and peptic digestion of labeled erythrocyte ghosts, the membrane-integrated segments of band 3 protein, the 17,000 and 10,000 dalton peptides, were isolated. At 2 mM initial label concentration, 90% of the hydrophobic probe phenylisothiocyanate was recovered in the 10,000 dalton peptide, the remaining amount of label being associated with the 17,000 dalton fragment. Pretreatment of the membranes with 5 mM p-sulfophenylisothiocyanate followed by labeling with 2 mM phenylisothiocyanate results in a consistent reduction in binding of phenylisothiocyanate by 1 mol/mol isolated band 3 protein. p-Sulfophenylisothiocyanate reportedly binds to the 17,000 dalton fragment (Drickamer, K. (1977), J. Biol. Chem. 252, 6909-6917). The interaction of the polar probe with the membrane protein affects binding of phenylisothiocyanate to the 10,000 dalton peptide by the equivalent of 1 mol/mol isolated peptide. The topological interrelation of the membrane-integrated segments is concluded.

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [125I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[14C]-cyanate binding to BR. [125I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.

Philicity of amino acid side-chains for photogenerated carbenes

Abstract The selectivity of a diazirine-photogenerated carbene towards amino acid side-chains was investigated by analysing amino acid retention following photocoupling with an immobilized carbene precursor. The heterobifunctional photocross-linker 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine was synthesized and coupled to fibre glass. Photoinduced amino acid binding to the solid support was analysed. The immobilized diazirine-photogenerated carbene preferentially binds to cysteine and aromatic amino acids. Amino acids carrying sulphur or oxygen as side-chain heteroatoms are, in general, more carbene-philic than amino acids with aliphatic side-chains. Marginal carbene insertion is obtained with glycine. On the basis of the empirically determined photocoupling capacities of the applied amino acids, a carbene philicity scale has been established. For homologous amino acids, carbene selectivity partly correlates with their hydrophobicity and the number of chemical bonds. Consequences of this distinct binding capacity are discussed with respect to photoselective protein modification.

Characterization of phenylisothiocyanate as a hydrophobic membrane label

Various labeling techniques have been widely employed to classify those portions ofmembrane proteins exposed to the aqueous environment [l-3]. With the recent introduction of hydrophobic azides, the lipophilic membrane-integrated domains became specifically accessible for chemical modification [4,5]. However, a marked disadvantage of the rapid, photoinduced reactivity of azides is that it is not exclusively directed towards selected functional groups of proteins [6]. Lipid components of the membrane system are quite highly labeled [7]. For specific modification of the hydrophobic moieties of membrane constituents group specific reagents are necessary. Successful modification in the apolar phase requires that the reagent be hydrophobic and partitions in favor of the apolar phase. Furthermore, the functional groups of the membrane protein must be in a reactive state. No less important, the reaction and reaction products formed are required to be nonionogenic and noncharged, respectively. Water should not be involved in the reaction, neither as reactant nor as product. PITC is able to fulfill these requirements. It reacts with ammo groups solely in the unprotonated form [8]. The reactivity of the protein functional groups can therefore be controlled by the pH in the aqueous phase. At neutral pH, the exposed protein amino groups are expected to be protonated. The buried, bulk pHindependent amino functions, however, can be in the reactive state. This implies selectivity of the reaction for non-aqueous systems.

Heterobifunctional crosslinking of bacteriorhodopsin by azidophenylisothiocyanate

Phenyliso~iocyanate has been described for group-specific modification of membrane proteins in the apolar membrane phase [I]. The method has been applied in various membrane systems [l-4]. Upon modification of purple membranes a single unique nucleophilic group of bateriorhodopsin (lysine 2 15) is labeled by phenylisothiocyanate [5]. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Therefore, the phenylisothiocy~ate modification is concluded to occur in the hydrophobic membrane domain. The newly described probe p-azidophenylisothiocyanate combines the group specificity of phenylisothiocyanate and the advantages of photoinduced reactivity of arylazides. Since both reagents react with proteins from within the lipid bilayer, the new probe possesses the requirements necessary for hydrophobic, heterobifunctional crosslinking. The reagent interacts in a first step with deprotonated proteinaceous nucleophiles. The erosslink is then formed by light activation of the hetero-function (arylazide). In this study the investigation of molecular characteristics of p-azidophenylisothiocyanate, the modification of bateriorhodopsin by the bifunctional probe, and the flash-induced crosslink-formation are described. The versatility of the group-specific, heterobifunctional probe is discussed.

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein I. Covalent modification and inhibition of phosphate transport

The hydrophobic probe phenylisothiocyanate is utilized for chemical modification of human erythrocyte band 3 protein. The binding of phenylisothiocyanate to this protein is characterized in whole erythrocytes, erythrocyte ghost membranes and in isolated band 3 protein. The label, reactive with nucleophiles in their deprotonated form is found in all three preparations to be covalently bound to band 3 protein. Under saturation conditions, 4--5 mol phenylisothiocyanate are covalently bound per mol protein (molecular weight 95 000). The described modification effects inhibition of phosphate entry into erythrocytes. 50% inhibition of phosphate transport is obtained following a preincubation of erythrocytes with 0.45 mM phenylisothiocyanate. Both phenylisothiocyanate binding and transport inhibition are saturating processes. The relationship of the two parameters is non-linear.

Immobilization of biomolecules on polyurethane membrane surfaces.

Lipophilic polymer membranes incorporating binding sites are widely used in various potentiometric, amperometric, and optical sensors. Here, we report on the biofunctional modification of the surface of a Ca(2+)-selective membrane. A photoactivatable biotin derivative was synthesized and covalently immobilized on a soft polyurethane membrane. The modified polymer was characterized by X-ray photoelectron spectroscopy (XPS) as well as by potentiometric measurements. The selective binding of streptavidin by the photo-cross-linked biotin derivative was demonstrated. The surface coverage obtained with different experimental protocols was analyzed by autoradiography using [(35)S]-streptavidin. The new approach may significantly extend the scope of applicability of potentiometric sensors.