Andreas Rosenberg

Evidence of an affinity threshold for IgE‐allergen binding in the percutaneous skin test reaction

Background Atopy is an aberrant immune response involving allergen‐specific IgE production, though serum IgE concentration is not an entirely reliable diagnostic tool, particularly for epidemiological and genetic studies. There is no clear correlation between IgE and other indicators of atopy such as skin prick tests (SPT)s, and physiological associations are difficult to justify in cases with detectable IgE but negative SPT results.

The activation of carnosinase by divalent metal ions

Abstract 1. 1. A cell and a procedure for the electrodialysis of protein solutions has been described which is suitable for the removal of stabilizing Mn 2+ ions from solutions of purified carnosinase. The rate of dialysis obtained is considerably higher than the rate of inactivation of the metal-ion free enzyme. 2. 2. It has been shown that Mn 2+ ions, besides stabilizing the enzyme, also act as activators, performing the role of a coenzyme for the enzymic reaction. 3. 3. Of the divalent metal ions investigated Cd 2+ , Mn 2+ , Zn 2+ and Co 2+ are shown to function as activators for carnosinase. The apparent efficiency of activation is given by the order: Cd 2+ > Mn 2+ ⪢ Zn 2+ > Co 2+ . 4. 4. The Mn 2+ activated enzyme is inhibited both by ions functioning as activators, such as Zn 2+ and Co 2+ , and by non-activating ions with Be 2+ and Fe 2+ as the most effective inhibitors. 5. 5. The time course of the enzymic reaction in the presence of Mn 2+ , Cd 2+ and Zn 2+ is shown to be of different type for each of the ions. The factors influencing the course of the reaction are discussed. 6. 6. The influence of the concentration of free Mn 2+ and Cd 2+ ions on carnosinase activity has been studied. The results are, in the case of Cd 2+ , in accordance with the assumption that one Cd 2+ ion interacts with each active site. The influence of Mn 2+ ions on the measured activity is most simply explained, by assuming the necessity for two metal ions per active site. It is suggested that the second Mn 2+ ion is necessary for stabilizing the enzyme molecule. 7. 7. The metal-ion specificity of carnosinase and its dependence on the properties of the metal ions and the conditions of the assay have been discussed.

Altered allergen binding capacities of Amb a 1-specific IgE and IgG4 from ragweed-sensitive patients receiving immunotherapy

BACKGROUND The mechanisms for the effectiveness of allergen immunotherapy (IT) are not well understood. The binding potential for immunoglobulins is a function of both antibody concentration and affinity (K(A)). PURPOSE The purpose was to perform a cross-sectional preliminary study to investigate any differences in allergen-specific antibody affinity and concentration following ragweed immunotherapy by introducing a new concept of antibody binding capacity ([Ig] X K(A)). METHODS The binding capacity of allergen-specific IgE and IgG4 was determined for ragweed-allergic individuals undergoing ragweed immunotherapy and compared with the capacity of ragweed-specific IgE and IgG4 for allergic individuals not receiving immunotherapy. RESULTS The mean binding capacity for IgG4 after long-term immunotherapy was 1.6 log units higher (P < .0001) than for individuals not receiving IT. The binding capacity for allergen-specific IgE was 1.2 log units lower following long-term immunotherapy (P < .0001) compared with individuals not receiving ragweed IT. CONCLUSIONS We hypothesize that a primary effect of immunotherapy is to increase IgG4 binding capacity and concomitantly decrease IgE binding capacity.

An automated method for determination of antibody affinity distribution functions with nanogram quantities

We have developed a method to determine the binding affinity distribution functions (the probability density function for affinity constants will be referred to as affinity distribution throughout the text) for human serum antigen specific IgE for their respective allergens. This fully automated method, based on the Access technology of Sanofi Diagnostics Pasteur, is a highly sensitive two-step sandwich immunoassay requiring only nanogram quantities of antibodies. Allergen-antibody binding isotherms can be determined covering a range of 5 magnitudes of ligand (allergen) concentration. An affinity distribution function, describing the polyclonal nature of the antibody response, can be calculated from the binding isotherm. The validity of the method is assessed using mAbs against the purified allergen Der p 1. The resolving power and sensitivity of the method are demonstrated using selected sera from donors with high and low levels of specific IgE for these allergens. We are able to show that heterogeneous IgE populations can be determined using 100 microl of sera containing 150 pg of specific IgE per ml. Overall, the range of affinities that can be determined by this method is 1 x 10(6)-1 x 10(11) M(-1).

Viscosity and transient solvent accessibility of Trp-63 in the native conformation of lysozyme

We have measured the rates of isotope exchange at the nitrogen of the indole ring of Trp-63 of lysozyme and of L-tryptophan as a function of solution viscosity. We have used two cosolvents, glycerol and ethylene glycol, to modify the relative viscosity. We have derived the appropriate kinetic equations for the alternative possibilities that the exchange takes place either in solution or in the intact protein matrix. Because we chose to study the proton-catalyzed exchange reaction, the rate of it is not expected to be diffusion-limited. We confirmed this by measuring the exchange from tryptophan. These results and the known effects of glycerol and ethylene glycol on the solvation of indole allow us to predict that if the exchange reaction takes place in a protein matrix the effects of the two cosolvents when compared under isoviscous conditions should be identical. This is what we find for Trp-63 in lysozyme at 15, 20 and 26 degrees C. The slope of the linear plot of log k vs. log relative viscosity is 0.6. This strongly supports a model for conformational fluctuations where transient solvation takes place without major changes in protein folding. The most interesting feature of our findings is the fact that a slow reaction admittedly not diffusion-limited shows, when taking place in a protein matrix, a linear dependence on solution viscosity. We suggest that what we observe is the effect of damping of movement of the side chain expressed as a change in the friction along the reaction coordinate in the corresponding phase space. The presence of such effects stresses the validity and usefulness of Kramers model of rate processes for reactions taking place in a protein matrix. Such behavior is predicted by several of the recently proposed general mechanisms of enzyme catalysis.

Optical rotatory dispersion of liver alcohol dehydrogenase, and its complexes with coenzymes and inhibitors

Abstract The optical rotatory dispersion of liver alcohol dehydrogenase (LADH), its binary complex with NADH 2 as well as that of the ternary complexes LADH-NADH 2 -isobutyramide and LADH-NAD-pyrazole, was determined in phosphate buffer, ionic strength 0.1 at pH 7.0. The measured values were used to calculate Moffitt-Yang plots for the different systems. The contributions from Cotton effects in the visible and ultraviolet region introduced by the binding of NAD or NADH 2 to the protein were estimated and expressed as correction terms. The change of Moffitt parameter b 0 from −100 ° for the enzyme to −185 ° in case of the ternary complexes was interpreted as a change of protein conformation due to the formation of the complex.

QUANTITATION OF [3H]THYMIDINE UPTAKE BY STIMULATED HUMAN LYMPHOCYTES†

We have investigated the relationship between cell numbers and the amount of tritiated thymidine ([3H]TdR) taken up by stimulated human peripheral lymphocytes, as a function both of labeling time and of the specific activity of the thymidine. Cells responding either to mitogens or to allogenic cells show simple first order kinetics for the uptake of thymidine. Fitting the data to a Michaelis‐Menten type of model, we observe for labeling times of 12 hr and longer, non‐competitive inhibition of thymidine uptake by increased specific activity of tritium label, regardless of the mode of stimulation. However, for an individual responder in MLC at any arbitrary but fixed specific activity, dose of [3H]TdR and labeling interval, we still observe a linear relationship between cell mass and incorporated label. In contrast, if specific responding combinations in mixed lymphocyte culture are compared, the inhibition by specific activity at longer time intervals becomes significant and influences the quantitative interpretation of results. Specific activities of less than 10 Ci/mmole and labeling times of 6 hr or less avoid inhibition and ensure a linear relationship between dividing cell number and CPM (counts per minute recorded) of incorporated label.

The affinity of allergen specific IgE and the competition between IgE and IgG for the allergen in Amb a V sensitive individuals

We have calibrated a solid state RAST assay with affinity purified allergen-specific IgE. We then utilized the calibrated assay to measure the average affinity of individual IgE-containing sera in terms of the average association constant < K > for purified allergen Amb a V. The binding data yielded linear reciprocal plots indicating that the range of affinities of the responding clones was narrow. The range of the average association constant for the IgE-Amb a V complex was 0.9-26 x 10(10) M-1. The average affinity of the corresponding IgG response in the same individual, estimated by inhibition studies of IgE binding, was 10(7) M-1 in one case and lower than 10(6) M-1 in all the other cases.

Gated quenching of intrinsic fluorescence and phosphorescence of globular proteins: an extended model

We present a theoretical model to account for the quenching data of macromolecular fluorescence and phosphorescence when the accessibility to the quencher is gated by a dynamic mechanism coupled to the fluctuation of the macromolecular matrix. We show that the model currently in use to interpret gated quenching processes gives only approximate results in both qualitative and quantitative terms, and it can be regarded as a specific case of the presented model. We show that the gating dynamics affect both the apparent accessibility (alpha obs) and Ksv values obtained by the modified Stern-Volmer plot. The effect of gating on alpha obs and Ksv depends upon the relative rate of gating compared to the excited state lifetime. The model allows us to predict the effect of viscosity on quenching if it takes place by a gated mechanism. The prediction can and is, in this case, compared to the existing data on glycerol effects on acrylamide quenching of the tryptophan fluorescence in RNAse T1. The result shows that a simple gated model is not compatible with the observed quenching behavior.

Interaction of acrylamide with proteins in the concentration range used for fluorescence quenching studies

Abstract 14 C labelled acrylamide was synthesized and used in equilibrium dialysis measurements to study the binding of acrylamide to the proteins: human serum albumin (HSA), ovalbumin and cod paryalbumin III. Our intent was to determine whether binding takes place in the concentration range that is used for the study of fluorescence quenching by acrylamide. In contrast to previously published reports, we found that all the proteins investigated did bind acrylamide. The affinity of this interaction, when interpreted in terms of multiple independent binding equilibria, was very low, K ass ⋍ 0.5–2 M −1 ; however, due to multiple binding sites, a considerable amount of acrylamide is to be found in the protein phase at concentrations used for quenching experiments. The number of binding sites seems to vary with the protein. A pH 7.0 the binding to the 69 kD HSA corresponds to 14 ± 8 sites, the binding to the 45 kD ovalbumin corresponds to 40 ± 25 sites, whereas for the 11 kD cod parvalbumin the binding corresponds to only a few sites. The binding is very sensitive to pH and to the presence of cosolvents such as glycerol. At pH 5.2, close to the isoionic point, the number of binding sites for acrylamide on HSA increases to > 100. The very weak binding justifies an alternative description of the phenomena as a distribution equilibrium between two phases. In such a model we see that the formal concentration of acrylamide in the protein volume is in some cases higher than in solution ( K eq = 1.2 for HSA at pH 5.2). These findings suggest that any model describing the quenching of fluorescence in proteins by acrylamide has to account for the presence of two pools of acrylamide and consequently for the presence of multiple modes of quenching.