Interaction of Proteins with Hydrogen Ions and Other Small Ions and Molecules

Steinhardt, Jacinto; Beychok, Sherman

doi:10.1016/b978-0-12-395724-5.50012-0

Cited by 50 publications

(44 citation statements)

References 271 publications

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“…Nb is the normality of acid or base, MP is the mass (g) of protein ( N × 6.25), and α is the average degree of dissociation of the α -NH 2 groups released during hydrolysis expressed as

\begin{matrix} α = \frac{10^{pH - p K}}{1 + 10^{pH - p K}}, \end{matrix}

where pH is the value at which the hydrolysis was conducted. The pK values were calculated using the equation below [22]

\begin{matrix} p K = 7.8 + \frac{298 - T}{298 T} \times 2400, \end{matrix}

where, T is the hydrolysis temperature in Kelvin. …”

Section: Methodsmentioning

confidence: 99%

Enzyme Hydrolysates fromStichopus horrensas a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides

Forghani

Ebrahimpour

Bakar

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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Stichopus horrens flesh was explored as a potential source for generating peptides with angiotensin-converting enzyme (ACE) inhibitory capacity using 6 proteases, namely alcalase, flavourzyme, trypsin, papain, bromelain, and protamex. Degree of hydrolysis (DH) and peptide profiling (SDS-PAGE) of Stichopus horrens hydrolysates (SHHs) was also assessed. Alcalase hydrolysate showed the highest DH value (39.8%) followed by flavourzyme hydrolysate (32.7%). Overall, alcalase hydrolysate exhibited the highest ACE inhibitory activity (IC50 value of 0.41 mg/mL) followed by flavourzyme hydrolysate (IC50 value of 2.24 mg/mL), trypsin hydrolysate (IC50 value of 2.28 mg/mL), papain hydrolysate (IC50 value of 2.48 mg/mL), bromelain hydrolysate (IC50 value of 4.21 mg/mL), and protamex hydrolysate (IC50 value of 6.38 mg/mL). The SDS-PAGE results showed that alcalase hydrolysate represented a unique pattern compared to others, which yielded potent ACE inhibitory peptides with molecular weight distribution lower than 20 kDa. The evaluation of the relationship between DH and IC50 values of alcalase and flavourzyme hydrolysates revealed that the trend between those parameters was related to the type of the protease used. We concluded that the tested SHHs would be used as a potential source of functional ACE inhibitory peptides for physiological benefits.

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“…Nb is the normality of acid or base, MP is the mass (g) of protein ( N × 6.25), and α is the average degree of dissociation of the α -NH 2 groups released during hydrolysis expressed as

\begin{matrix} α = \frac{10^{pH - p K}}{1 + 10^{pH - p K}}, \end{matrix}

where pH is the value at which the hydrolysis was conducted. The pK values were calculated using the equation below [22]

\begin{matrix} p K = 7.8 + \frac{298 - T}{298 T} \times 2400, \end{matrix}

where, T is the hydrolysis temperature in Kelvin. …”

Section: Methodsmentioning

confidence: 99%

Enzyme Hydrolysates fromStichopus horrensas a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides

Forghani

Ebrahimpour

Bakar

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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“…Ions can bind to charged residues in a pH-dependent manner at low ionic strength thus screening the charge. The binding is nonlinear with salt concentration (Steinhardt & Beychok, 1964;von Hippel & Schleich, 1969;Record et al, 1978). Assuming that there are n identical binding sites for a ligand L on the tetramer at a given pH and any ions bound to the monomer are not dissociated on tetramer formation, we can define a perturbed equilibrium:…”

Section: Effect Of Ionic Strengthmentioning

confidence: 99%

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

Wilcox

Eisenberg

1992

Protein Science

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The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an a-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 "C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pK, values of 7.5 and 8. [7][8][9][10][11][12][13][14][15][16][17][18] agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 "C depending on the pH, unfolding above and below that temperature. AC; for folding ranges from -0.085 to -0.102 cal g" K-', comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). A H o and ASo are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, w., 1959, Adv. Protein Chem.14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 "C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 "C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system. Abbreviations: CD, circular dichroism; far UV, 260-185 nm; near UV, 330-245 nm; AzgO, light absorption at 280 nm; AC:, the standard change in heat capacity from the unfolded to the folded form; At = AL -AR, where AL and AR are the absorbance of left and right circularly polarized light, respectively; AcZU, Ac at 222 nm; AtZB2, At at 282 nm; Th, a temperature at which AHo(T) = 0; T,, a temperature at gates to form an a-helical tetramer. The amount of tetwhich ASo( T ) = 0; TES, N-tris(hydroxyrnethyl)methyl-2-aminoethane sulfonic acid. Keywords

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“…The magnitudes and positions of the difference maxima near 230 mp clearly indicate that in some cases at least changes of other kinds must contribute. The most likely of these, depending on the protein and the conditions are: (a) changes in the ionization state of histidine residues, a frequent consequence of denaturation under some circumstances [21,22]; (b) an a-helix -> random coil transition, which is accompanied by a diminution in inten- Table 2. Difference spectral characteristics of model compounds and proteins L1 and L 2 refer to the two near-ultraviolet maxima in the difference spectra and S to the short-wavelength maximum.…”

Section: Fig 7 Absorption Spectra Of N-acetyltyrosine Ethyl Ester Imentioning

confidence: 99%

An Analysis of Perturbations in the Ultraviolet Absorption Spectra of Proteins and Model Compounds

Bailey¹,

Beaven

Chignell³

et al. 1968

European Journal of Biochemistry

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It is shown that the perturbation of the ultraviolet absorption spectra of the tyrosine and tryptophan chromophores in aqueous solutions by addition of solvents of lower polarity leads not only to red shifts but also to changes in peak intensity, and oscillator strength. The effect is much greater in tyrosine than in tryptophan, and can be as large as 30°/, (transfer from water to ethanol). It is shown that observed difference spectra can be reasonably reproduced by the addition of two calculated curves, one corresponding to a shift, the other to a change in intensity, i.e. an admixture of shift difference spectrum and absolute spectrum. Similar intensity changes are seen to accompany the denaturation of several proteins. The changes of the characteristic parameters of difference spectra with the concentration of perturbant are analyzed. The relation between the difference spectra generatcd by aromatic chromophores near 230 mp and those in the 280 mp region are considered, and their relative magnitudes have been estimated for a variety of perturbants. It is shown that in denaturation of a number of proteins, the difference spectra near 230mp cannot be satisfactorily accounted for solely in terms of the model compounds containing aromatic chromophores. Possible contributions to difference spectra of proteins in this region are considered.Ultraviolet difference spectra have been widely used to follow conformational processes in proteins [1,2]. The shifts in the tyrosine and tryptophan spectra are relatively small (of the order of 1 mp) and the bands are broad. It is in consequence impossible to make any reasonably precise quantitative estimation of the shift, neither is it possible to use it per se for the quantitation of conformational changes, since in an isomerisation a parameter is required which will reflect the relative concentrations of the two states, i.e. an absorbance difference. For this reason, it is usual to use difference spectrophotometry, in which in effect a shift is converted into an absorbance difference.Other factors than a shift can, however, influence the shape and magnitude of the difference spectrum, in particular broadening or sharpening of the vestigial vibrational structure which casn be seen in tyrosine and tryptophan spectra, and also changes in total intensity. Although these factors may have been recognized, no attempt has been made to analyse their contribution to observed effects in proteins. An examination of published difference spectra does indeed reveal the presence of considerable and unexplained differences in shape, and it is our intention to analyse these in greater deta.il.We shall consider also the difference spectrum generated in proteins on denaturation, centred near 230 mp [3], and its relation to the more familiar effects in the 280 mp region. MATERIALS AND METHODS MaterialsL-Tyrosine, DL-tryptophan and their methyl ester hydrochlorides were obtained from Sigma Chemical Co., and were chromatographically pure. Urea was several times recrystallized from ethanol until it...

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Interaction of Proteins with Hydrogen Ions and Other Small Ions and Molecules

Cited by 50 publications

References 271 publications

Enzyme Hydrolysates fromStichopus horrensas a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides

Enzyme Hydrolysates fromStichopus horrensas a New Source for Angiotensin-Converting Enzyme Inhibitory Peptides

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

An Analysis of Perturbations in the Ultraviolet Absorption Spectra of Proteins and Model Compounds

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