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Biomedical subjects

J R Cann

Publications and source records attributed to J R Cann.

At least 55 records · Page 3Linked to original sources
PubMed1985

Effects of diffusion on the electrophoretic behavior of associating systems: the Gilbert-Jenkins theory revisited.

The Gilbert-Jenkins theory predicts the asymptotic shape of moving-boundary sedimentation and electrophoretic patterns and broad zone molecular sieve chromatographic elution profiles for the class of interacting systems, A + B in equilibrium C, in which two dissimilar macromolecules react reversibly to form a complex. A particularly provocative case is the one in which the complex has a greater migration velocity than that of either reactant, each of which has a different velocity. Depending upon conditions, this case predicts, for example, that in the asymptotic limit an ascending electrophoretic pattern or a frontal gel chromatographic elution profile can show two hypersharp reaction boundaries separated by a plateau. This prediction is now confirmed by numerical solution of transport equations which retain the second-order diffusional term and extrapolation of the computed patterns to zero diffusion coefficient. For finite diffusion coefficient, however, the two hypersharp reaction boundaries are separated by a weak negative gradient. These calculations are extended to an examination of the transitions between the three types of patterns admitted by the case under consideration in order to gain physical understanding and to define criteria for recognizing the transitions. Studies of this kind not only establish confidence in the Gilbert-Jenkins theory, but, in addition, they provide new insights which make for more effective application of the theory to real systems.

Diffusion↗
PubMed1985

Theory of counterion electrophoresis. Guidelines for determination of ligand-binding parameters.

A theory is formulated to provide guidelines for the quantitative interpretation of steady-state counterion electrophoretic patterns (T.-H. Ueng and F. Bronne, Arch. Biochem. Biophys. 197 (1979) 205) in terms of intrinsic ligand-binding constant and number of binding sites on the protein molecule. Briefly, the prescribed procedure calls for extrapolation of the steady-state binding constant to infinite dilution of protein to obtain a quantity which is the product of a readily evaluated kinetic factor and the intrinsic binding constant. On the other hand, extrapolation of the steady-state number of binding sites to infinite dilution can probably be dispensed with if determined at a reasonably low protein concentration.

Calcium-Binding Proteins↗
PubMed1984

Electrophoresis of reacting systems involving sulfhydryl oxidation of proteins.

Numerical solution of the relevant continuity equations has been used to examine the possible effects of intramolecular sulfhydryl oxidation on the electrophoresis of proteins. Simulations of moving boundary electrophoresis, based on variants of a previous model [J.R. Cann, N.H. Fink, and D.J. Winzor (1983) Arch. Biochem. Biophys. 221, 57-63], show that the Schlieren patterns for the ascending and descending limbs are likely to exhibit pronounced nonenantiography. Whereas the pattern for one limb may comprise essentially a single peak, that for the conjugate side can exhibit bimodality, the nature of which is time dependent. Bimodality of the Schlieren pattern can develop in either the ascending or descending limb of the electrophoresis cell, depending basically upon the number of sulfhydryl groups available for oxidation, and on the relative magnitudes of the rate constants describing the oxidation and the isomerization of the oxidized protein species. Whether the faster-moving or slower-moving peak grows with time is shown to depend upon the magnitude of the electrophoretic mobility of the resultant isomer in relation to that of the oxidized protein species. Schlieren patterns for fish muscle creatine kinase and rabbit muscle aldolase are then used to support the relevance of these predictions to moving boundary electrophoresis of proteins undergoing intramolecular sulfhydryl oxidation. Finally, numerical simulation of the zonal electrophoretic behavior of such systems serves to illustrate that bimodal patterns may also obtain, thereby giving a false impression of inherent protein heterogeneity. Emphasis is therefore placed on the importance of maintaining an adequate concentration of reducing agent throughout the medium in which the protein migrates, a potential problem in polyacrylamide gel electrophoresis at neutral pH.

Chemical Phenomena↗
PubMed1983

Theory of electrophoresis of reacting systems as applied to the sulfhydryl oxidation of creatine kinase.

In an attempt to explain the unusual electrophoretic behavior of fish muscle creatine kinase, a phenomenological theory of transport of reacting systems has been formulated for the electrophoresis of a sulfhydryl protein undergoing oxidation in the presence of a gradient of molecular oxygen. The model assumes slow O2-oxidation of sulfhydryl groups followed by rather rapidly reversible sulfhydryl-disulfide interchange with concomitant change in the electrophoretic mobility of the protein. The computed electrophoretic patterns for this model exhibit a sharp, unimodal ascending boundary but a bimodal descending boundary in which the proportions of the two peaks are time dependent. There is a striking similarity between the theoretical patterns and their experimental counterparts (M.D. Doherty, D.A. Bergman, V.M. Re-Miller, and D.J. Winzor, 1980, Arch. Biochem. Biophys. 202, 558-564); and hence support for consideration of the electrophoresis of fish muscle creatine kinase in these terms.

Animals↗
PubMed1983

A combined spectroscopic study of the solution conformation of bradykinin.

CD, NMR and laser Raman spectroscopy have been used in a complementary fashion to study the conformation of bradykinin and related peptides. The results are interpreted to mean that the secondary structure of bradykinin in aqueous solution is a time average of two interconverting structures - one disordered and the other partially ordered. The peptide evidently spends a maximum of about 20% of its time in the partially ordered conformation which features a hydrogen-bonded structure at the C-terminus, most likely a gamma-turn with Pro7 as the second residue. Bradykinin and its analogs enjoy considerable conformational adaptability in terms of response to changes in temperature and solvent composition. Thus, dioxane favors the hydrogen-bonded structure, while aqueous NaCl0(4) and methanol favor the trans Pro7 isomer. The pronounced CD changes caused by increasing the temperature are attributed to a conformational transition, which is reflected in the CD of the Phe residues and possibly also the Arg-Pro-Pro moiety. The magnitude of the thermodynamic functions for the presumed transition are interpreted to mean that the structure of water plays a dominant role in determining the conformation of the peptide.

Amino Acid Sequence↗
PubMed1982

Theory of sedimentation for ligand-mediated heterogeneous association-dissociation reactions.

Theoretical analytical sedimentation patterns have been computed for ligand-mediated heterogeneous association-dissociation reactions between macromolecules. Involvement of either a single kind of ligand or two different ligands acting in a stepwise fashion has been considered. Self-association, mediated in a stepwise fashion by two different ligands, has also been examined. The conclusion reached is that such interactions have the potentiality for exhibiting as many as three or four sedimenting peaks despite rapid rates of reaction. In general, the peaks correspond to different equilibrium compositions and not to individual macromolecular species; that is to say, they constitute a reaction boundary. Their resolution depends upon generation of concentration gradients of ligand(s) along the centrifuge cell by chemical reequilibration during sedimentation of the several macromolecular species. The implications of these findings for fundamental studies on subunit proteins and protein assemblies and for conventional applications of ultracentrifugation are discussed.

Kinetics↗
PubMed1980

Determination of ligand-binding constants by isoelectric focusing. Generalization and extension of theory.

The theory of a proposed isoelectric focusing procedure for the determination of ligand binding constants has been generalized (via numerical calculations) to non-linear, electrophoretic velocity profiles and systems for which the change in velocity of the protein accompanying ligand binding depends upon pH. The theory has also been extended to include binding of ligand to heterogeneous sites. Determination of accurate values of the binding parameters depends upon extrapolation of apparent parameters to infinite dilution of protein.

Journal Article↗
PubMed1979

Circular dichroism study of the solution conformation of luteinizing hormone releasing hormone.

A systematic investigation has been made into the circular dichroic behavior of luteinizing hormone releasing hormone and its peptide fragments and deletion analogues. The results are interpreted to mean that the hormone exists in solution as an ensemble of conformers with different sensitivities to temperature and solvent composition. The far-ultraviolet circular dichroic spectra exhibited by the hormone under different experimental conditions can be simulated satisfactorily by the weighted addition of the spectra of its aliphatic- and aromatic-containing halves. However, the structure of the hormone is not simply the sum of its halves, since some conformational feature of the intact molecule perturbs the near-ultraviolet circular dichroism of its aromatic residues.

Amino Acid Sequence↗
PubMed1979

Isoelectric focusing of interacting systems. IV. interaction of macromolecules with each other and with ligands.

The theoretical isoelectric focusing behavior for rapidly reversible, bimolecular complexing between two macromolecules depends upon the relative value of the isoelectric point of the complex. When it is intermediate in value, the transient patterns exhibit three peaks. As equilibrium is approached the central peak of complex disappears leaving two reactant peaks. When the isoelectric point is acidic or alkaline to both reactants, the equilibrium pattern also shows two peaks; but in this case only one is pure reactant, the other being a reaction zone. The two cases can be distinguished by varying the relative amounts of reactants. Transient patterns for ligand-binding exhibit a peak of unliganded protein and a reaction zone. As the charged ligand is driven out of the focusing column the reaction zone disappears, so that the equilibrium pattern shows only a peak of unliganded protein. In general, the isoelectric point of the complex cannot be determined from the transient patterns.

Journal Article↗
PubMed1979

Isoelectric focusing of interacting systems. V. Determination of ligand-binding constants.

A theory is formulated for an isoelectric focusing procedure which permits determination of intrinsic ligand-binding constants. The protein is first focused in the absence of ligand, after which ligand is added to the appropriate electrode compartment and then driven by the electric field into the focusing column where it complexes with the protein. The band of protein and its complexes moves to the constituent isoelectric point. An equation linearly relates the reciprocal of the overall distance moved to the reciprocal of the local concentration of ligand. The quotient of the intercept and slope gives the intrinsic binding constant. If the concentration of ligand in the electrode compartment is used in lieu of the local concentration, an apparent constant is obtained. Extrapolation of the apparent constant to infinite dilution of protein gives the intrinsic constant. For certain systems, conditions may be realized which give an apparent constant within 4% of the intrinsic constant.

Journal Article↗
PubMed1979

Effect of temperature upon the circular dichroism of bradykinin.

Analysis of the effect of temperature on the circular dichroism spectrum of bradykinin has led to a more precise understanding of the solution conformation of the peptide. Circular dichroism and 13C n.m.r. have been used in a complementary fashion to support the picture that bradykinin spends a maximum of about 20% of its time in a partially ordered conformation featuring a gamma-turn with Pro7 as the second residue. Since the gamma-turn probability is insensitive to temperature, some other conformational effect dominated by the structure of water presumably produces the pronounced change in the circular dichroism spectrum with increasing temperature.

Bradykinin↗