PubMed · 4702463
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J A O'Connell. 1973. Improving titration accuracy.. https://pubmed.ncbi.nlm.nih.gov/4702463/
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The mass spectra of several compounds with molecular weights in the 2500-20,000 Da range were obtained with a quadrupole mass spectrometer equipped with an atmospheric pressure ion source. Average molecular weight determinations of mellitin (2846.4 Da), a synthetic oligonucleotide (4262.8 Da), myoglobin (16,950.4 Da) and on the subunits of beta-lactoglobulin (18,277.1 Da) requiring as little as 1 pmol of material were achieved with accuracies and precisions of +/- 1 Da. An ion-spray interface was used to produce ions via the ion evaporation process, producing mass spectra containing a series of multiply-charged molecular species. A simple method for calculating the molecular weight of unknown compounds from the spectra containing multiply-charged ions is described.
Results obtained from gel filtration showed that beta-lactoglobulin binds p-nitrophenyl phosphate with a stoichiometry of 1 mol of ligand per 18,360 monomer. Circular dichroic spectra confirmed the binding and implicated tryptophan and phenylalanine residues in the interaction. Fluorescence of the protein was quenched on binding also supporting complex formation; analysis of these data indicates that p-nitrophenyl phosphate binds to beta-lactoglobulin A with a dissociation constant of 31 microM. The B and C genetic variants of beta-lactoglobulin bind p-nitrophenyl phosphate with dissociation constants of 63 and 70 microM, respectively. In addition, a series of other nitrophenyl compounds and pyridoxal phosphate were also investigated by fluorescence analysis and found to bind to the protein. These results are discussed with respect to a recent hypothesis that beta-lactoglobulin binds retinol and is structurally related to serum retinol binding protein.
If sedimentation equilibrium and sedimentation velocity experiments are performed on a self-associating solute under the same solution conditions, it is possible to evaluate the sedimentation coefficients (si) of the self-associating species and the usual concentration dependence parameter (g or gs). We have tested some of these methods with simulated examples. A more critical test is to use real data. Sedimentation equilibrium experiments with beta-lactoglobulin A at 20 degrees C in 0.2 M glycine buffer (pH 2.46) indicated that a nonideal monomer-dimer association was present. Sedimentation velocity experiments were performed on beta-lactoglobulin A under the same conditions. Using data from both sets of experiments we were able to evaluate s1, s2, g and gs using two different models for swa, the apparent weight average sedimentation coefficient. The empirical model for swa developed by Weirich et al. [1] gave better variance than did the model for swa developed by Gilbert and his co-workers [2-5]. Using a simulated monomer-dimer association mimicking a system having higher sedimentation coefficients than beta-lactoglobulin A did, we were able to show that one could not obtain s2 from tangents to the plot of 1/swa vs. c in the high concentration region. The methods developed here for sedimentation coefficients can be applied to other experiments in which a weight average property (or its apparent value) of a self-associating solute is measured, provided the appropriate thermodynamic experiments are done under the same solution conditions.