Complete Titration Research Articles

Spectroscopic Studies on the Conformation of Cytochrome c and Apocytochrome c

Abstract The appearance of the downfield region of the PMR spectrum of apocytochrome c is consistent with that of an extensively disordered protein. The resonances of the three histidine C-2 protons are almost equivalent and have a pKa of 6.2. In contrast, this region of the ferricytochrome c PMR spectrum shows many sharp resonances, due to the tertiary structure of the protein and contact shifts from the heme group. The complete titration of one histidine residue, with a pKa of 6.41, can be determined in the holoprotein. At low pH, the resonance of a second histidine residue can be observed. The acid-induced conformational transition of ferricytochrome c can be resolved into several component transitions by the use of different spectral parameters. Measurements of absorption at 395 nm and fluorescence emission at 340 nm show a single cooperative transition with midpoint of about pH 2.5. These indices are closely related to the geometry of the heme group. The resonances of the two observable histidine residues change in a way suggesting denaturation only below pH 1.8 but indicate conformational changes at about pH 3. The resonances assigned to heme methyl groups, however, undergo significant changes between pH 3 and 4. The reduced viscosity of acid-denatured ferricytochrome c and the fluorescence emission of its tryptophan residue increase markedly as salt concentration is lowered, indicating that the conformation of the acid denatured protein is sensitive to ionic strength.

The determination of sulphate in sea water by means of photometric titration with hydrochloric acid in dimethyl sulphoxide

The sulphate concentration in sea water has been determined by photometric titration to hydrogen sulphate in dimethyl sulphoxide solution with hydrochloric acid, using bromocresol green as indicator. The end-point is evaluated graphically and a complete titration can be performed in less than 15 min. Since borate, carbonate and hydrogen carbonate interfere, a separate determination of the alkalinity is necessary. The method can be used satisfactorily for sea water samples with a chlorinity of at least 2‰.

Potentiometric titration of the fluorescence yield of spinach chloroplasts

The fluorescence yield of spinach chloroplasts was measured as a function of oxidation-reduction potential under anaerobic conditions at pH 6.0, 7.0, and 8.0. The potentiometric-titration curve showed two fluorescence quenching processes, both quenching in the oxidized state. The complete titration curve could be reasonably fit with a Nernst equation for the sum of two one-electron-transfer reactions with different midpoint potentials. At pH 7.0 the two transitions had midpoint potentials of about −35 mV and −270 mV and both showed a pH dependence of about −60 mV per pH unit. The hypothetical quencher, Q, assumed to be responsible for light-induced fluorescence-yield changes in green photosynthetic systems could be ascribed to the more positive quenching component.

The sulfhydryl groups of muscle phosphorylase. I. Number and reactivity.

The sulfhydryl groups of glycogen phosphorylase from rabbit muscle have been investigated with respect to their reactivity with p-chloromercuribenzoate (PCMB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, N-ethylmaleimide (NEM), iodoacetic acid, and iodoacetamide. By spectrophotometric titration, PCMB reacts with 6 of the 18 half-cystine residues in native phosphorylase b, and with 12–14 of the 36 half-cystine residues in native phosphorylase a, and causes complete inactivation in both cases. Reaction of approximately 6 more residues in phosphorylase b (12 in phosphorylase a) occurs when the protein is denatured with HCl or sodium dodecyl sulfate (SDS).DTNB, cystine, and iodoacetic acid react with only two sulfhydryl groups in native phosphorylase b, and do not cause any loss of activity. Iodoacetamide can also react with two sulfhydryl groups without causing any loss of enzymic activity, but activity is lost in proportion to the extent of titration of the next four groups. DTNB reacts with 12 groups in SDS-denatured phosphorylase b but with only 9 groups in urea- or guanidine HCl-denatured enzyme. NEM and iodoacetic acid react with 14 groups in urea-denatured phosphorylase b. Two experiments are reported that provide additional confirmation of previous reports of the absence of disulfide bridges in phosphorylase.We conclude that two sulfhydryl groups per mole of phosphorylase b can react with any reagent without loss of enzymic activity. When the correct reagent and conditions are chosen, a further class of four sulfhydryl groups reacts, resulting in complete loss of enzymic activity. Thus, only two specific sulfhydryl groups on each monomer unit need be titrated to inactivate glycogen phosphorylase and cause it to dissociate. Upon denaturation of phosphorylase by several of the usual protein denaturants, another class of sulfhydryl groups reacts readily, the exact number depending on the reagent and the conditions, but the complete titration of all 18 sulfhydryl groups per molecule of phosphorylase b occurs only under special conditions. These results are of general relevance to procedures employing various protein denaturants and sulfhydryl reagents for the titration of protein sulfhydryl groups.

Semi-automatic determination of calcium and magnesium hardness in water

A photometric method is described for the determination in a single titration of both calcium and magnesium hardness in waters using only one indicator. The method involves the addition of Eriochrome Black T to the solution containing calcium and magnesium buffered to pH 10, followed by titration with EDTA. The course of the titration is followed on a simple semi-automatic apparatus which is described. The complete titration curve obtained from a potentiometric recorder shows two distinct end-points; this permits the determination of both calcium and magnesium from a single titration.

Molecular changes in myosin caused by methylmercuric hydrome

An ultracentrifugal study has been made on myosin treated with methylmercuric hydroxide. 1. 1. Myosin is transformed by tritration of its -SH groups with methylmerucuric hydroxide to a faster-sedimenting product. This occurs in the same range of methyl-mercuric hydroxide titration at which the calcium-activated ATPase activity is inhibited. A parallelism can be demonstrated between the loss of ATPhase activity and the loss of myosin due to the transformation process. 2. 2. Complete titration of the -SH groups of myosin with methylmercuric hydroxide causes the release of a small subunit with sedimentation rate of 1.6 S, at 25° but not at 15°. 3. 3. These results suggest that the -SH groups whose titration causes inactivation of ATPase activity are responsible for the configurational integrity of the active site, and that release of the small subunit is not related to titration of these -SH groups.

CHANGES IN THE BUFFER SYSTEM OF THE WHEAT PLANT DURING ITS DEVELOPMENT

In an earlier paper (5) it was reported that progressive changes occur in the acidity of the wheat plant during its seedling and maturation stages. Subsequent studies involving the complete titration curve have given a more comprehensive idea of the changes which take place in the titratable compounds of the juice. The results show a remarkably ordered relation between the buffer capacity of the juice, its hydrogen-ion and titratableacid concentration, and the stage of development of the plants.