L. Clem Gruen

Interaction of amino acids with silver(I) ions.

The reactivity of silver(I) ions towards twenty amino acids has been studied in aqueous unbuffered solutions using an ion-selective electrode as a highly sensitive monitor. Contrary to general belief, silver ions are not completely specific for cysteine, but also react with lysine, arginine, methionine and, to a minor extent, cystine. Nevertheless the interaction with all of these species except cysteine is too weak to affect significantly the determination of thiol in proteins by the customary argentometric titration method. The possible origin of errors arising in such titrations is discussed.

Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase

We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAGTM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of ∼4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its1H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.

Identification of legumin-like proteins in wheat

We have obtained several amino acid sequences from purified polypeptides of a wheat endosperm storage globulin previously described as ‘triplet’ protein. The amino acid sequence data supported by immunochemical analysis using anti-oat 12S globulin antibodies, provide definitive evidence that the triplet protein is homologous to pea legumin and related seed storage proteins of oats, rice and several dicotyledonous species. Thus, it is now proposed that the triplet protein of wheat be renamed ‘triticin’. The oat globulin antibodies also cross-reacted strongly with the high-molecular-weight (HMW) glutenin subunits which have been implicated in bread-making quality.

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti‐idiotype Fab′

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti‐idiotype antibody 3‐2G12 has been used as a model system to demonstrate experimentally the influence of non‐ideal binding effects on BIAcoreTM binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5×105 M−1 s−1), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site‐specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (KD) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the KD values obtained using the BIAcore

Stability and physicochemical properties of a trypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L)DC).

The stability and physicochemical properties of the major trypsin inhibitor from the winged bean seed (designated trypsin inhibitor 2) have been studied in solution. The purified inhibitor, which stoichiometrically inhibits bovine trypsin in the molar ratio 1:1, is stable over the pH range 3-11 at ambient temperatures. Only a slight decrease in inhibitory activity occurs down to pH 2, but a sharper decrease occurs at pH values above 11. The inhibitor is stable to heat up to 60 degrees C, but at higher temperatures (60-90 degrees C) it is more stable at pH than at pH 5.5 or pH 8.0. Trypsin inhibitor 2 retains its inhibitory activity in 8 M urea at pH 8.0, but is more susceptible to 8 M urea at pH 4.0. The stronger denaturant 6 M guanidine hydrochloride, however, abolishes the inhibitory activity at both pH 4.0 and pH 8.0. The inhibitor was not inactivated in 0.14 M beta-mercaptoethanol at either pH; however, reduction in the presence of 8 M urea or 6 M guanidine hydrochloride results in a loss of inhibitory activity. Circular dichroism and optical rotatory dispersion studies indicate that the inhibitor structure is characterized by beta-sheet and unordered forms and the absence of alpha-helix. The positive CD band centered at 227 nm has been used to follow conformation change as a function of temperature. In line with the stability studies, the inhibitor conformation was thermally most stable at pH 3.0 and changed increasingly as the pH was raised. This band showed little change at neutral pH up to 8 M guanidine hydrochloride. Tyrosine titration in aqueous solution indicates that 1 or 2 of the 11 tyrosines are difficult to titrate even at pH 13. A more normal titration curve is obtained in 6 M guanidine hydrochloride, although at least one tyrosine side-chain appears to be buried in the protein interior and resists complete titration at pH values in excess of 12. These data show that this inhibitor has a high degree of stability, typical of other known protein proteinase inhibitors.

Quantitative analysis of the interaction between lysozyme and monoclonal antibody D1.3.

The method of sedimentation equilibrium has been used to determine the stoichiometry and binding constant for the interaction between hen egg white lysozyme and monoclonal antibody D1.3. The procedures described allow the relative binding affinities of 125I-labelled lysozyme and unlabelled lysozyme to be compared. The data indicate that labelled and unlabelled lysozyme bind to monoclonal D1.3 with similar affinity (binding constant, K = 1.6 x 10(9)/M). Using solid-phase methods estimates obtained for the binding constant were lower and dependent both on the amount of antigen coated on the plate and the dilution of primary antibody (D1.3). These data were not consistent with a simple equilibrium binding model, suggesting kinetic or orientation effects. In contrast sedimentation equilibrium experiments provide a rapid and accurate method for determining both the stoichiometry and binding constants for the interaction between antigens and antibodies.