Maria Spinetti

New method for separation and determination of denatured caseins by hydrophobic interaction chromatography

A new method for the separation of denatured alpha-, beta- and kappa- caseins by hydrophobic interaction chromatography (HIC) is proposed. The method is based on an easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate (GdmSCN) and elution on a TSK-Gel(R) Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea in the mobile phase. The procedure, applied to commercial caseins and to real, raw samples (whole milk powder and fat-free yoghurt) is not expensive, it requires common high performance liquid chromatography (HPLC) instrumentation and allows the separation of caseins also in the presence of whey proteins. Quantitative results on the analysis of alpha-, beta- and kappa-caseins in real samples are also reported.

Radiochromatographic Method for –SH Determination in Proteins by [203Hg] p-Hydroxymercuribenzoate

A radiochromatographic method which allows the evaluation of the number of sulfhydryl and disulfide groups per molecule of protein, in native or denatured (reduced or not) form, in terms of –SH groups, is proposed. The experiments were performed by using 203Hg-labelled p-hydroxymercuribenzoate as reagent and known concentrations of bovine serum albumin (BSA) were used as a standard. The method is precise with a mean RSD of 3.0, 6.5 and 2.5% for native, denatured and reduced BSA, respectively; the accuracy is in the range 1.9–5.0%.

203Hg labelled PHMB as reagent for the determination of -SH groups in native and denatured proteins by hydrophobic interaction chromatography

Aldolase, glyceraldehyde 3-phosphate dehydrogenase and ovalbumin were determined by hydrophobic interaction chromatography, which allows the evaluation of the number of –SH groups per molecule of protein in both the native and denatured form. These proteins were chosen as models to show the generality of use of the procedure for analytical and biochemical applications. The experiments were performed by using 203Hg-labelled p-hydroxymercuribenzoate as reagent and known concentrations of proteins. The results were compared with FI-CV-ETAAS measurements and literature data.

Substitution reactions at palladium(II) complexes producing dimeric species: stability constants determination for simultaneous equilibria

Substitution equilibria in Pd(dien)-like aquocomplexes are parallel to dimerization equilibria of the starting monomeric species. Accurate potentiometric measurements of the hydrogen ion concentration lead to the simultaneous determination of the stability constants of both kinds of equilibria: Pd(dien)H2O2+ + X− ⇄ Pd(dien)X+ + H2O, and 2 Pd(dien)H2O2+ + X− ⇄ [Pd(dien)]2X3+ + 2H2O. For the monomeric complexes, the following relative stability series was ascertained: X− = Cl− < Br− < I− < SCN− < OH−. The bridging ligand affects the relative stability of the dimeric species in the order: X− = I− ⪡ OH− < SCN−.

Simultaneous determination of two serine proteinases by hydrophobic interaction chromatography

SummaryThe possibility of determining more than one enzyme at the same time has been examined. A new approach, based on the measurement of a direct and specific chromatographic signal obtained by hydrophobic interaction chromatography (HIC) of the stable complex formed with the inhibitor aprotinin, is proposed. A basic procedure for the quantitative determination of trypsin and kallikrein, taken as models, is described. The method is precise with a mean coefficient of variation of 3.1% and 3.5% for trypsin and kallikrein, respectively; the limit of determination for both enzymes is 0.17 nmol ml−1 in the original sample.

Hydrophobic interaction chromatography separation and determination of glycolytic enzymes-method validation

SummaryA new method, based on the measurement of a direct and specific chromatographic signal obtained by hydrophobic interaction chromatography of some glycolytic enzymes from rabbit skeletal muscle, is presented. The enzymes examined were: aldolase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, 3-phosphoglyceric phosphokinase, enolase, phosphoglucomutase, phosphoglucose isomerase, L-lactate dehydrogenase, pyruvate kinase. The possibility of simultaneous determination of six enzymes in a synthetic mixture has been tested. In this approach to the quantitation of such enzymes the use of the substrate is avoided: the method eludes the measurement of enzymatic activity, utilised only for check purposes, and allows direct determination in terms of molarity. For each enzyme, reproducibility, linearity range, recovery ≥97%) and detection limit are reported. The method has been applied to three commercial crudes: by a simple procedure, the identification of more enzymes has been possible and the simultaneous determination of some of them has been performed. For such enzymes recoveries were quantitative and their determination is of good precision with a mean coefficient of variation 2.43–3.0%.

125I-labelled aprotinin as reagent for simultaneous determination of serine proteinases by hydrophobic interaction chromatography

SummaryA previously described method for determining more than one serine proteinase simultaneously by hydrophobic interaction chromatography of their complexes with aprotinin is inapplicable when other UV-absorbing species are co-eluted. The suitability of125I-labelled aprotinin has therefore been tested as a reagent in the analysis of mixtures containing trypsin, α-chymotrypsin and kallikrein taken as models, in the presence of ribonuclease and lysozyme. A new procedure is described which, without introducing changes in the chromatographic separation, allows direct determination of serine proteinases in terms of molarity. Results obtained in experiments with solutions containing from 0.20 to 30.00 nmol of each serine proteinase are reported.

Purification of125I-labelled aprotinin by hydrophobic interaction chromatography

Summary125I-aprotinin has been prepared and purified by affinity chromatography in combination with hydrophobic interaction chromatography (HIC). The method allows the preparation of the labelled inhibitor practically carrier-free, in saline solution without organic modifier and the isolated compound retained inhibition activity. Recovery of radioactive material was virtually quantitative, without substantial accumulation of radioactivity on the column. Compared with currently used procedures, the method is more reliable because the labelled polypeptide is collected by following its specific chromatographic signal.

Direct determination of kallikrein by high-performance liquid chromatography

A direct and specific identification of porcine pancreatic kallikrein by high-performance hydrophobic chromatography is proposed; the minimum amount which can be injected is 2.5 U. An application to the quantitative determination of the enzyme by high-performance size-exclusion chromatography is reported; the method is precise with a mean coefficient of variation of 2.8% and the minimum amount which can be injected is 0.02 U of kallikrein. The results obtained with determinations in real biological samples (porcine pancreatic powder and human urine) are reported. The method is based on direct and specific chromatographic signals and does not destroy the biological activity of this enzyme.

Determination of aprotinin by titration with bovine trypsin with end-point detection by high-performance liquid chromatography

A method for the determination of aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is described. The procedure involves the formation of the BPTI-trypsin complex in the presence of an excess of BPTI, quantitative separation of the residual BPTI from the mixture by affinity chromatography and identification and evaluation of the residual BPTI by reversed-phase high-performance liquid chromatography. The method is precise with a mean coefficient of variation of 4.0 and 4.3% for intra- and inter-assay runs, respectively, and has a limit of determination of 3.0 micrograms of aprotinin. The proposed method can be applied to commercial samples, even in very dilute solutions, for the standardisation of aprotinin.