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A I White

Publications and source records attributed to A I White.

4 recordsLinked to original sources
PubMed1977

Proton magnetic resonance studies of histidines in human, rhesus monkey, and bovine carbonic anhydrases.

Histidine C-2 proton resonances in rhesus monkey carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) and bovine carbonic anhydrase were investigated using 270-MHz proton magnetic resonance. The results suggest that there are extensive three-dimensional homologies between the human B and rhesus B enzymes and between the human C and bovine enzymes. Resonances from solvent exchangeable protons have been observed in the 11-16 ppm range in the NMR spectra of human carbonic anhydrases B and C and bovine carbonic anhydrase. Up to five of these are sensitive to changes of pH and the presence of inhibitors. Three of these resonances are assigned to NH protons of the metal coordinated imidazole groups. These results are discussed in relation to various models for the catalytic mechanism of carbonic anhydrase.

Animals↗
PubMed1976

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...

Animals↗
PubMed1975

Antibody--hapten interactions in solution.

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.

Amino Acids↗