Binding domains of colicins E1, E2 and E3 in the receptor protein BtuB of Escherichia coli.

Eight reagents specifically modifying amino acids were applied to cells of a standard Escherichia coli colicin indicator strain to follow in vivo changes of its binding capacity for colicins E1-E3 and hence the binding domains (epitopes) for them in the outer membrane receptor protein BtuB. The effect of these reagents was also investigated in a mutant strain carrying an extensive BtuB deletion. The following differences of the binding epitopes could be ascertained. Colicin E1: Blockage of OH-groups, just as N-substitution of His and modification of Arg and Trp enhance binding of colicin E1. In the deleted receptor, also abolition of carboxylic anion bonds enhances its affinity for colicin E1. It follows that colicin E1 is bound, most of all, to the hydrophobic domain A (loops 1 + 2) of BtuB. Colicins E2 and E3: both exert rather analogous binding parameters. In contrast to E1, O-substitution of Ser and Thr dramatically decreases the E2 and E3 binding, similarly to modification of Lys. There is also a clear difference in the binding affinity of the domain for E2 and/or E3 and for E1 following modifications of their Arg and His. Colicins E2 and E3 are rather bound to the hydrophilic domain B (loops 5-7) of the receptor. In this respect, interactions of colicins E2 and E3 with deeper parts of A and B domains (Trp, several Arg, Lys and His residues) exhibited subtle differences. Acidic pH (4.5-6.0) shows a positive, while pH 7.0-8.5 a rather negative impact on the receptor-binding function for the colicins. It was clearly demonstrated that there is just a partial difference between the binding behavior of colicins E1, E2 and/or E3.

Sensitive amperometric biosensor for the determination of biogenic and synthetic amines using pea seedlings amine oxidase: a novel approach for enzyme immobilisation.

We prepared a new inorganic sorbent based on modified triazine (2-[4,6-bis (aminoethylamine)-1,3,5-triazine]-Silasorb; BAT-Silasorb) which binds pea seedlings amine oxidase (PSAO) very tightly without loss of its catalytic activity. This unique feature as well as the wide substrate specificity of PSAO was successfully utilised in the construction of an amperometric biosensor based on a carbon paste electrode for the fast and sensitive detection of various amines at a formal potential 0 mV versus Ag/AgCl reference electrode. The reaction layer of the biosensor is created by the direct immobilisation of PSAO at the electrode surface via affinity carrier BAT-Silasorb. Used arrangement facilitates a simple restoration of the inactive biosensor. An amperometric signal results from horseradish peroxidase catalysed reduction of H2O2, a secondary product of the oxidative deamination of amines, catalysed by PSAO. The sensor was used for the basic characterisation of 55 biogenic and synthetic amines, from numerous mono-, di- and polyamines to various hydroxy-, thio-, benzyl- and aromatic derivatives in order to establish its suitability as a postcolumn detector. Its high sensitivity to putrescine 20.0 +/- 0.64 mA l-1 per mol (636.9 +/- 2.03 mA l-1 per mol per cm2), a limit of detection of 10 nmol l-1 (determined with respect to a signal-to-noise ratio 3:1), a linear range of current response to 0.01-100 mumol l-1 concentration of substrate and good reproducibility all indicate that the sensor could be applied to future industrial and clinical analyses.

Improved chromatographic purification of pea seedlings diamine oxidase.

An improved and simplified purification procedure has been developed for the isolation of diamine oxidase from pea seedlings (DAO EC 1.4.3.6). It involves ammonium sulphate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography and size-exclusion chromatography. The homogeneity of the final enzyme preparations and molecular weight were determined by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE). The isoelectric point of 7.35 +/- 0.05 was determined by chromatofocusing and by polyacrylamide gel isoelectric focusing.

Amperometric glucose biosensor with extended concentration range utilizing complexation effect of borate.

Borate buffer strongly decreases amperometric response of a glucose oxidase linked pO2 or H2O2 sensing electrode, extending substantially its linear calibration range. With increasing pH and concentration of the buffer the upper limit for glucose can be varied between 1 and 30 mmol l-1 glucose. The effect of borate ion is explained by the rapid complexation of glucose decreasing the equilibrium concentration of free beta-anomer, the specific substrate of glucose oxidase. The high loading of cross-linked enzyme inside the sensor membrane is necessary for the measurement to ensure an almost constant response factor (delta i per 1 mmol l-1) between pH 5 and 10. Analysis in stirred solution and in a flow-through system has been employed for the measurement of elevated glucose levels in heparinized human blood or plasma samples.

The effects of some protein-modifying reagents on the interaction of colicins A, E2, E3, and K with their respective Escherichia coli cell receptors.

Colicins attach themselves--through specific protein-protein interactions--onto receptors in the outer membrane of sensitive bacterial cells. An attempt was made to analyze amino-acid groups and attractive forces involved in this interaction, following treatment of either colicins A, E2, E3 and K or sensitive bacteria with various physico-chemical factors and several protein-modifying reagents. The amounts of colicin bound were checked by a quantitative biological assay. Ionic conditions and specific spatial conformation of both colicin and its receptor molecules are crucial in their interaction and, hence, in the biological effect of colicin. Formaldehyde, naphthalene-diisocyanate and osmium tetroxide strongly inhibit the binding ability of all colicins tested. The results suggest that NH2 and SH groups are involved in their binding onto receptors; also, CH3S groups seem to be engaged in the attachment of colicins E2 and E3 and phenol-OH groups in that of colicin K. The possible involvement of further groups (NH, SH etc.) should be checked using more specific reagents. The attitude of colicins E2 and E3 to their common receptor Btu B protein is nearly, but not completely the same. Receptors for all colicins tested should be oxidized to achieve optimal interactions; obviously, carbonyl groups are produced and newly formed anions increase the negative load of bacterial surface. In agreement, reduction of at least colicins A and E3 enhances their receptor binding reactivity. The binding capacity of each receptor can be modulated by a set of amino acid reagents in a specific manner.

Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor.

Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.