Normal Titration Research Articles

Fluorescent derivatives of nucleotides. Metal ion interactions and pH dependency

The fluorescence parameters of ethenoadenosine derivatives are influenced by metal cations and pH, as summarized here. The pH profile of ethenoadenosine determined by fluorescence intensity gives a normal titration curve and is not affected by ionic strength. In contrast, the pH titration curves of etheno-ATP, etheno ADP, and etheno AMP depend upon ionic strength. At high ionic strength normal curves are obtained, whereas at low ionic strength anomalies are obtained; this suggests that the phosphates can interact with the ring, possibly by hydrogen binding to the ring nitrogens. The room temperature fluorescence of ethenoadenosine occurs from the base form, although excitation of either the acid or base forms can contribute to the emission. This result can be explained if the excited state pK is lower than the ground state pK, and if deprotonation occurs within the time scale of the excited state. At low pH values the fluorescence lifetime of the base form is dependent upon the buffer concentration, indicating that the reverse reaction, protonation, occurs. The affinity constants for the binding of metals to the ethenoadenosine phosphates resemble those for the corresponding adenosine phosphates. Ni(II) and Co(II) are more effective than Mn(II) in quenching the fluorescence of ethenoadenosine phosphates; this result is predicted by Förster's theory for energy transfer based upon the overlap between donor emission spectrum and acceptor absorption spectrum. The diamagnetic ions Mg(II), Ca(II), and Zn(II) do not appear to affect the fluorescence of the ethenoadenosine phosphates directly, but rather to affect the conformation of the molecule, thereby affecting the quantum yield.

Buoyant titration of ovalbumin in four alkali halides. Hydration and ion binding

The buoyant titrations of ovalbumin in CsCl, RbCl, CsBr and RbBr were measured between pH 2 and 13. The buoyant densities were found to depend on the salt employed to generate the density gradient and the solution pH. At low pH, nearly identical buoyant densities were observed in solutions having a common anion while at high pH, salt solutions having the same cation produced nearly indentical buoyant densities. The buoyant density of ovalbumin in RbBr was found to decrease as the pH was increased from 2 to 6. This is the first demonstration of a drop in the buoyant titration curve for a biopolymer.A model based on an invariant partial specific volume, normal titration of all amino acid residues, the binding of a counter-ion to each ionized residue and all hydration associated with these salt pairs was constructed and satisfactorily accounted for all four buoyant titration curves. These results indicate that no water is bound to the neutral portion of this protein.

The charge-relay system of serine proteinases: Proton magnetic resonance titration studies of the four histidines of porcine trypsin

Peaks corresponding to the C (2)-protons of all four histidine residues of porcine β-trypsin were resolved in 250 MHz nuclear magnetic resonance spectra after deuteration of the slowly exchangeable N- H groups (whose resonances obscure the histidine peaks) by reversible unfolding of the protein in D 2O. One of the four peaks was assigned to the charge-relay histidine in the active site of trypsin (His(57) in the bovine chymotrypsinogen numbering system). Whereas the three other histidine C (2)-peaks exhibited normal titration curves with single p K′ values of 7.20, 6.71 and 6.67, the peak assigned to His(57) had an abnormal titration curve showing two protonation steps in the pH range from 1 to 9. The first protonation with a pH′ mid of 5.0 is rapid on the nuclear magnetic resonance time-scale; the second with a pH′ mid of 4.5 is slow and apparently involves conformational transitions between two states having lifetimes of approximately 18 ms. In the complex between porcine β-trypsin and bovine pancreatic trypsin inhibitor (Kunitz) His(57) was found to be insensitive to pH over the range from 4 to 9 and its chemical shift resembles that of His(57) in the singly protonated charge relay of free trypsin. This result provides direct evidence that the trypsin charge relay acts as a proton acceptor in the initial catalytic step which leads to the formation of a tetrahedral complex. In the presence of equimolar bovine pancreatic trypsin inhibitor (Kunitz) the pH' mid of the conformational transition that affects the charge-relay histidine is lowered from 4.5 to approximately 3.5.

Steroid–acid colour reactions. Part I. Carbon-13 nuclear magnetic resonance spectra of various protonated aliphatic ketones and their pKBH+values

The 13C n.m.r. spectra of cyclopentanone, diethyl ketone, di-isopropyl keton cyclohexanone, dicyclopropyl ketone, cyclopropyl methyl ketone, and cyclohex-2-enone in sulphuric acid of varius concentrations have been recorded. The maximum downfield change in the chemical shift, the apparent effect of complete protonation of the carbonyl group, is ca. 30 p.p.m. A plot of chemical shift against acidity shows that there is a gradual increase in apparent protonation but the form is not that of a normal titration curve. Solvation of the protonated ketone is suggested as explanation. A plot of log I against –H0 has a slope of ca. 0.3. Values of H0 at apparent halfprotonation are reported and 13C n.m.r. is shown to be an excellent method for the determination of the apparent basicity of a ketone. The results are treated by the method of Bunnett and Olsen and values of the thermodynamic pKBH+ are obtained. The protonation of steroids is discussed.

Proton magnetic resonance spectra or porcine muscle adenylate kinase and substrate complexes.

Porcine muscle adenylate kinase with a molecular weight of 22,000 has 2 histidine, 5 phenylalanine, 7 tyrosine, and no tryptophan residues. The effect of pH, substrate, and the paramagnetic manganous ion on the proton magnetic resonance spectrum of the enzyme, particularly the aromatic region, has been investigated at 220 MHz. The well resolved C2 proton peaks of the 2 histidine residues have been individually assigned to His-36 and His-189 by comparison with the spectrum of the carp muscle enzyme which has only one C2 proton peak and only 1 histidine residue, 36. The chemical shift of the peak designated C2-H of His-36 in the porcine enzyme has a normal titration curve with a pKalpha = 6.3 but the peak for His-189 is not titratable in the pH range 5.8 to 8.1. The pKalpha of the single His-36 of the carp enzyme is similar to that of His-36 of the porcine enzyme. Changes in pH, particularly at low pH, also affect the chemical shifts of the tyrosine residues. Occupation of either the monophosphate site by AMP or the triphosphate site by ATP or GTP causes a downfield shift of the C2-H of His-36, and the equilibrium mixture causes an even greater shift, but no shift in the C2-H of His-189. The substrates also induce changes in the chemical shifts in the phenylalanine-tyrosine region of the spectrum. Tentative assignments of the highest and lowest field peaks in this region have been made based on the three-dimensional structure determined by x-ray crystallography. On the basis of these assignments, it is concluded that Phe-183 is unperturbed by substrate binding but that Tyr-153 or -154 at the hinge of the molecule, are perturbed. The C2-H of adenine and C8-H of adenine or guanine of the bound substrates were also observed; those of AMP are unperturbed but C2-H of ATO is shifted downfield and the C8-H of ATP and GTP are shifted upfield. The paramagnetic manganous ion had no effect on the spectrum at Mn(II) to enzyme ratios below 1:10; above this ratio, a general broadening was observed...

Drug–Biomolecule Interactions: Proton Magnetic Resonance Studies of Complex Formation between Bovine Neurophysins and Oxytocin at Molecular Level

Proton magnetic resonance spectroscopy was used to monitor individual amino acid residues in bovine neurophysin, in the nonapeptide hormone oxytocin, and in the complex formed between them. For neurophysin I alone, a normal titration curve for the C‐2 proton resonance of the lone histidine residue was obtained with an apparent ionization constant of 6.9. Addition of oxytocin to a solution of neurophysin I at pH 6.5 resulted in several changes in the spectrum. The effect on the histidine C‐2 proton resonance signal indicated a slow exchange process between two states, probably representing a conformational change in the protein. The apparent pK of the histidine residue in the hormonal complex was shifted to 6.7, indicating a slightly more positive (less electron dense) environment for the histidine residue. Resonances of the single tyrosine residue of oxytocin were observed to broaden significantly, but not to shift appreciably, on the addition of neurophysin II. These observations may indicate involvement of the tyrosyl residue of oxytocin in the hormone—“carrier protein” interaction.

Determination and interpretation of the differential titration curves of proteins: The differential curves of bovine methemoglobin, CO‐hemoglobin and serum albumin

AbstractIn this paper a refinement of the usual experimental titration procedure of proteins is described, by means of which the differential titration curve can be obtained with good accuracy. From this curve the positions of the inflection points of the normal titration curve can be localised with better precision, and, as is shown, the analysis of the differential curve has certain advantages above that of the normal curve. The capabilities of experimental and analysing procedure are illustrated by results, obtained with the proteins bovine methemoglobin (MetHb), bovine CO‐hemoglobin (HbCO) and bovine serum albumin (BSA).

Acid–base properties of fibers. Part II. A polyelectrolyte theory of the combination of fibers with acids and bases

AbstractA. new theory for the acid–base properties of fibers is presented, resembling those for polyelectrolytes, and based on the normal titration of the charged groups of the fibers and the energy needed to remove the protons from the fiber to the solution, against the electrostatic, osmotic, and affinity differences. The acid titration equation for a poly‐ampholyte fiber is: where pK0′ is the intrinsic dissociation constant of the titration groups in the fiber, α is the degree of dissociation, χ is the electrostatic potential of the fiber, τ the osmotic pressure, V̄H the partial molar volume of the hydrogen ion, and Δμ the difference of standard chemical potential in the fiber and the solution. In all cases examined, the osmotic term proved to be negligible. The electrostatic potential was calculated as suming it to be uniform over the fiber. For wool, pK0′ is independent of salt concentration but varies linearly with degree of dissociation, indicating that two pK's are operating, pKq = 3.585 for paired groups and pKp = 4.855 for nonpaired groups at 0°C., present in equal proportions. Titrations of wool with sulfuric acid and a wide range of strong monobasic acids also obey the theory, and values of the anion affinities in general agreement with those given by Gilbert and Rideal are found. The acid titration of native ox‐hide collagen gave a constant value of pK0′ = 4.0. For nylon, the value of pK0′ is, to a first approximation, independent of degree of dissociation and salt concentration, in agreement with the theory.

Precipitin Testing with Special Reference to the Photoelectric Measurement of Turbidity

Summary The photoelectric measurement of turbidities (by use of the Libby photron-reflectometer) developing in the precipitin reaction has been found to be correlated with the nitrogen contents of the precipitates formed in protein antigen-antibody systems, and with the total amount of antigen and antibody precipitated in the case of the SIII-antibody system. Antisera prepared in rabbits to purified SIII, beef albumin, beef globulin II, pumpkin seed globulin, and Panulirus argus serum have been tested. The amounts of turbidity developed are decreased with increased NaCl concentration or with increased pH within the limits tested. Increased reactivity of beef globulin II followed Bloor's mixture extraction of the dried reagent. In all cases the effects of environmental factors on the reaction, or chemical treatments of the antigens, are predominantly shown in the zone of antigen excess, and the chief implication of this knowledge is that the whole or normal titration curve of constant antiserum with varied antigen from prozone to postzone is the minimal knowledge upon which valid conclusions relating to precipitin testing may be based. Many reasons are given as to why the use of such normal titration curves is advantageous, since their determination by photoelectric turbidimetry is accurate and relatively easy under conditions described herein. The errors due to fragmentary comparisons of single points or portions of curves, which may even be noncomparable ones, will thus be avoided.

A revision of the atomic weight of thallium

Since T. W. Richards in 1888 first devised the method of nephelometric titration and applied it to the precise determination of the ratios of soluble halides to silver, it has been the mainstay of atomic weight determinations in all countries. This is proof enough of the great advance he thus made in the accurate measurement of the fundamental ratios of chemistry. Yet, valuable as the method of nephelometric titration has been, it is, as many who have used it will admit, both extremely tedious and liable to uncertainties much greater than would appear from the results which are published. Despite the most careful observance of the conditions laid down by Richards and subsequent workers, it is a common experience to find that even after standing for the normal time, 7-14 days, a liquor showing on the first test approximate equivalence of silver and chloride, will, on adding small quantities of precipitant behave quite erratically, so that in extreme cases addition of chloride may, in subsequent tests, produce apparently an increase in dissolved silver, and vice versa. Thus, even where nephelometric tests proceed normally, there is always a fear that apparent equivalence of silver and halide may be illusory; and a considerable experience of such tests has given us the impression that in a normal titration, involving 5-10 grams of silver in, say, 4-6 litres of solution, the resulting uncertainty in the weight of silver is of the order of 0•0005-0•0002 gm. It is, further, no slight disadvantage that a nephelometric titration can hardly be completed in less than 2 1/2-3 weeks and usually extends over a period of 4-6 weeks. One of the principal objects of the present work has been, therefore, to devise and test a method of titrating halides with silver which shall be more rapid and precise. A good trial of any such method is afforded by the determination of the atomic weight of a heavy element of small valency, for which, evidently, the ratio of the halide to silver is unfavourable to the attainment of precise results. It was in part for this reason that the case of thallium was chosen. Apart from any question of method, moreover, there was good reason for a new determination of this atomic weight.