Use of reversed phase high pressure liquid cromatography for the physicochemical and thermodynamic characterization of oxyresveratrol/β-cyclodextrin complexes

Knowledge of the complexation process of oxyresveratrol with β-cyclodextrin (β-CD) under different physicochemical conditions is essential if this potent antioxidant compound is to be used successfully in both food and pharmaceutical industries as ingredient of functional foods or nutraceuticals, despite its poor stability and bioavailability. In this paper, the complexation of oxyresveratrol with natural CDs was investigated for first time using RP-HPLC and mobile phases to which α-, β-, and γ-CD were added. Among natural CDs, the interaction of oxyresveratrol with β-CD was more efficient than with α- and γ-CD. The decrease in the retention times with increasing concentrations of β-CD (0–4 mM) showed that the formation constants (K_F) of the oxyresveratrol/β-CD complexes were strongly dependent on both the water–methanol proportion and the temperature of the mobile phase employed. However, oxyresveratrol formed complexes with β-CD with a 1:1 stoichiometry in all the physicochemical conditions tested. Moreover, to obtain information about the mechanism of the oxyresveratrol affinity for β-CD, the thermodynamic parameters ΔG°, ΔH° and ΔS° were obtained. Finally, to gain information on the effect of the structure of different compounds belonging to the stilbenoids family on the K_F values, the complexation of other molecules, resveratrol, pterostilbene and pinosylvin, was studied and compared with the results obtained for the oxyresveratrol/β-CD complexes.     

Evaluation of the accessible inclusion sites in copolymer materials containing β-cyclodextrin

The accessible inclusion sites of insoluble copolymers containing β-cyclodextrin (β-CD) were studied in aqueous solutions by measuring the absorbance changes (decolourization) of phenolphthalein (phth) at pH 10.5. The various copolymers were reacted at different β-CD:crosslinker mole ratios with five individual types of crosslinker agents (epichlorohydrin (EP), sebacoyl chloride (SCL), terephthaloyl chloride (TCL), glutaraldehyde (GLU), and poly(acrylic) acid (PAA), respectively). The decolourization provided estimates of the 1:1 binding constants (K₁) for the β-CD monomer/phth complex. Comparable values of K₁ were measured for copolymer/phth complexes with highly accessible β-CD inclusion sites as compared with the 1:1 β-CD/phth complex. The surface accessibility of the β-CD inclusion binding sites for the polymers ranged from ∼10 to 72%. The observed variability of the inclusion sites was attributed to: (i) steric effects in the annular hydroxyl region of β-CD, (ii) the degree of crosslinking of the copolymer and (iii) the accessibility of the micropore sites within the copolymers. The Gibbs free energy (ΔG°) and site occupancy (θ) of phth adsorbed to the copolymer materials was estimated independently using the Sips isotherm model. The ΔG° values ranged between −27.6 and −30.9 kJ mol⁻¹ for the copolymers and are in close agreement with the value for the 1:1 β-CD/phth complexes (ΔG° = −27 kJ mol⁻¹) in aqueous solution.     

Thermodynamics of complexation of lanthanides by 3- and 4-hydroxybenzoic acids

The thermodynamic parameters (log β₁₀₁, ΔH₁₀₁, and ΔS₁₀₁) for the formation of the 1:1 complexes between lanthanide cations and 3- and 4-hydroxybenzoate anions were determined by potentiometric and calorimetric titrations in aqueous solutions of 0.10 M (NaClO₄) ionic strength at 25 °C.     

Inclusion complex of 2-chlorobenzophenone with cyclomaltoheptaose (β-cyclodextrin): temperature, solvent effects and molecular modeling

A thermodynamic study of the inclusion process between 2-chlorobenzophenone (2ClBP) and cyclomaltoheptaose (β-cyclodextrin, β-CD) was performed using UV–vis spectroscopy, reversed-phase liquid chromatography (RP-HPLC), and molecular modeling (PM6). Spectrophotometric measurements in aqueous solutions were performed at different temperatures. The stoichiometry of the complex is 1:1 and its apparent formation constant (K_c) is 3846 M⁻¹ at 30 °C. Temperature dependence of K_c values revealed that both enthalpy (ΔH° = −10.58 kJ/mol) and entropy changes (ΔS° = 33.76 J/K mol) are favorable for the inclusion process in an aqueous medium. Encapsulation was also investigated using RP-HPLC (C18 column) with different mobile-phase compositions, to which β-CD was added. The apparent formation constants in MeOH–H₂O (K_F) were dependent of the proportion of the mobile phase employed (50:50, 55:45, 60:40 and 65:35, v/v). The K_F values were 419 M⁻¹ (50% MeOH) and 166 M⁻¹ (65% MeOH) at 30 °C. The thermodynamic parameters of the complex in an aqueous MeOH medium indicated that this process is largely driven by enthalpy change (ΔH° = −27.25 kJ/mol and ΔS° = −45.12 J/K mol). The results of the study carried out with the PM6 semiempirical method showed that the energetically most favorable structure for the formation of the complex is the ‘head up’ orientation.     

The thermodynamics of the complexation of lanthanides by 1-hydroxy-4,7-disulfo-2-naphthoic acid

The thermodynamic parameters, log β, ΔH and ΔS, for formation of lanthanide-1-hydroxy-4,7- disulfo-2-naphthoic acid complexes have been determined at 25 °C in 0.10 M NaClO₄ solutions by potentiometric and calorimetric titrations. Under the experimental conditions, the data can be explained with the formation of LnL⁻, LnL₂⁵⁻ and LnHL complexes (H₂L²⁻ = 1-hydroxy-4,7-disulfo-2- naphthoic acid anion). At pH < 3 the LnHL complex is the major species, whereas by increasing pH the formation of LnL_n³⁻⁴ⁿ complexes becomes more important. The data are compared to the comparable data for complexing by aromatic carboxylic acids.     

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K_b, ΔG_b, ΔH_b, ΔS_b and ΔC_p) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC_p in a single experiment. We ruled out specific interactions of Na⁺ and Cl^- with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K_b, ΔG_b and ΔH_b to become less favorable, while ΔS_b became less unfavorable. From the variation of K_b with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔG_b at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH_b compared to 2PG (by 19-24 kJ mol^-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC_ps were negative, and less negative with increasing ionic strength. ΔC_ps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.     

Diastereomeric preference in 1,4,7-tris((S)-2-hydroxy-3-phenylpropyl)-1,4,7-triazacyclononanelithium(I) and its sodium(I) analogue

In N,N-dimethylformamide (DMF), 1,4,7-tris((S)-2-hydroxy-3-phenylpropyl)-1,4,7-triazacyclononane forms metal complexes, [M(S-thppc9)]⁺, for which log K (dm³ mol⁻¹)=3.01, 2.65, 2.66, 2.65, 2.42 and 7.59 (all±0.05) where M⁺=Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺ and Ag⁺, respectively. Variable temperature ¹³C{¹H} NMR spectroscopy shows that the interchange between equivalent forms of a single diastereomer occurs for [Li(S-thppc9)]⁺ and [Na(S-thppc9)]⁺ characterised by: k=43±5 and 2900±100 s⁻¹, at 298.2 K, ΔH^‡=22.5±1.6 and 33.8±1.6 kJ mol⁻¹, and ΔS^‡=−133±5 and −59±6 J K⁻¹ mol⁻¹, respectively. Gas phase ab initio modelling shows these complexes and their K⁺ analogue to preferentially form distorted trigonal prismatic Λ, Δ, and Λ diastereomers, respectively.     

Lysophosphatidic acid and lipopolysaccharide bind to the PIP2-binding domain of gelsolin

The binding of the gelsolin P2 peptide (residues 150-169) with lysophosphatidic acid (LPA) and lipopolysaccharide (LPS) was investigated by isothermal titration calorimetry. P2 binds to LPS with higher affinity than to LPA. For the interaction of 1-oleoyl-LPA with P2 in the absence of salt, K_d and ΔH° were 920 nM and −2.07 kcal/mol, respectively, at pH 7.4 and 25 °C. For the interaction of lipopolysaccharide (LPS) from P. aeruginosa with P2 under the same conditions, K_d was 177 nM and ΔH° was −7.6 kcal/mol.     

Interaction Mode between Inclusion Complex of Vitamin K3 with γ- Cyclodextrin and Herring-Sperm DNA

Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K₃(K₃), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K₃ inclusion complex. The results from ultraviolet spectroscopic method indicated that VK₃ and γ-CD formed 1:1 inclusion complex, with the inclusion constant K_f = 1.02 × 10⁴ L/mol, which is based on Benesi–Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K₃ and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K ^θ_25°C = 2.16 × 10⁴ L/mol, and K^θ_37°C = 1.06 × 10⁴ L/mol. The thermodynamic functions of the interaction between γ-CD-K₃ and DNA were Δ_rH_m^θ = ?2.74 × 10⁴ J/mol, Δ_rS_m^θ = 174.74 J·mol^?1K^?1, therefore, both Δ_rH_m^θ (enthalpy) and Δ_rS_m^θ (entropy) worked as driven forces in this action.     

Solid-state structures and thermal properties of inclusion complexes of the organophosphate insecticide fenitrothion with permethylated cyclodextrins

The X-ray crystal structures and thermal stabilities of the inclusion complexes formed between the organophosphate insecticide fenitrothion [O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate] and the host compounds TRIMEA and TRIMEB (permethylated α- and β-cyclodextrins, respectively) are reported. In the complex (TRIMEA)₂·fenitrothion 1, the guest phosphate ester group is disordered and the molecule is fully encapsulated within a novel TRIMEA dimer in which the secondary rims of the two host molecules are in close contact. In contrast, the complex TRIMEB·fenitrothion 2 is monomeric and the guest molecule is statistically disordered over two positions, with the phosphate group inserted in the host cavity in both cases. Thermal analysis indicated gradual and partial loss of the guest in 1 during heating between 130 °C and the melting point of the complex (∼200 °C), whereas complex 2 displayed significant mass loss only after fusion of the complex at 161 °C.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

Kinetic and equilibrium studies of cyclomalto-octaose (γ-cyclodextrin)-methyl orange inclusion complexes

Measurements of the equilibrium and temperature-jump u.v., visible, and induced c.d. spectra of Methyl Orange (MO) in the presence of cyclomalto-octaose (γ-cyclodextrin, γ-CD) have been carried out. Three mechanistic steps were detected through the temperature-jump data (25.0°):

where K₁, K₂, and K₃ are 45 (±7), 2.0 (±1.1) × 10⁶, and 6.1 (±2.5) × 10³ dm³.mol^?1, respectively, k₂ = 9.4 (±5.1) × 10⁹ dm³.mol^?1.s^?1, and k_?2 = 4.8 (±0.8) × 10³ s^?1. The equilibrium u.v./visible data are also consistent with this reaction scheme. The high stability of the dimer inclusion complex (MO)₂ · γ-CD compared to that of the monomer inclusion complex MO · γ-CD appears to be related to the annular diameter of γ-CD and demonstrates a degree of selectivity in cyclodextrin inclusion complexes. The (MO)₂ · (γ-CD)₂ complex also contains a dimer, included by both γ-CD molecules.     

Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- but not of β-, γ- and ζ-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only α-CAs showed esterase/phosphatase activity, whereas enzymes belonging to the β-, γ- and ζ-classes were completely devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a much better substrate for several human/murine α-CA isoforms (CA I, II and XIII), with k_cat/K_M in the range of 2681.6–4474.9 M^?1 s^?1, compared to 4-nitrophenyl phosphate (k_cat/K_M of 14.9–1374.4 M^?1 s^?1).     

Effect of cyclodextrins on the reactivity of fenitrothion

The hydrolysis reaction of fenitrothion was studied in water containing 2% dioxane and in the presence of native cyclodextrins (α-, β- and γ-CD) and two commercially available modified derivatives, namely, permethylated β- and α-cyclodextrin (TRIMEB and TRIMEA, respectively). The kinetics of the reaction in the presence of TRIMEA could not be measured because the complex formed is insoluble and precipitated even at low concentration. On the other hand, the reaction is only weakly affected by the presence of α-CD. The hydrolysis reaction is inhibited by all the other cyclodextrins. From the kinetic data the association equilibrium constants for the formation of the 1:1 inclusion complexes were determined as 417, 511 and 99 M⁻¹ for β-CD, TRIMEB and γ-CD, respectively. Despite the differences in the association constants for β- and γ-CD, the observed inhibition effect is about the same and this is due to the fact that the rate of hydrolysis in the cavity of γ-CD is smaller than that in the cavity of β-CD. The strongest inhibitor is TRIMEB and this result is consistent with the known structure of the complex in the solid state.     

Synthesis and characterisation of ferrocene-containing β-diketonato complexes of rhodium(I) and rhodium(III)

The synthesis of new β-diketonato rhodium(I) complexes of the type [Rh(FcCOCHCOR)(CO)₂] and [Rh(FcCOCHCOR)(CO)(PPh₃)] with Fc=ferrocenyl and R=Fc, C₆H₅, CH₃ and CF₃ are described. ¹H, ¹³C and ³¹P NMR data showed that for each of the non-symmetric β-diketonato mono-carbonyl rhodium(I) complexes, two isomers exist in solution. The equilibrium constant, K_c, which relates these two isomers in an equilibrium reaction, are concentration independent but temperature and solvent dependent. Δ_rG, Δ_rH and Δ_rS values for this equilibrium have been determined and a linear relationship between solvent polarity on the Dimroth scale and K_c exists. The relationship between RhP bond lengths, d(RhP), and ³¹P NMR peak positions as well as coupling constants ¹J(³¹P¹⁰³Rh) has been quantified to allow calculation of approximate d(RhP) values. Variations in d(RhP) for [Rh(RCOCHCOR′)(CO)(PPh₃)] complexes have also been related to the group electronegativities (Gordy scale) of the terminal β-diketonato R groups trans to PPh₃. A measure of the electron density on the rhodium centre of [Rh(RCOCHCOR′)(CO)(PPh₃)] may be expressed in terms of the IR carbonyl stretching wave number, ν(CO), the sum of the group electronegativities of the R and R′ groups, (χ_R+χ_R′), or the observed pK_a^′ values of the free β-diketones RCOCH₂COR^′. An empirical relationship between ν(CO) and either pK_a^′ or (χ_R+χ_R′) has also been quantified.     

Formation of a wrapped DNA-protein interface: experimental characterization and analysis of the large contributions of ions and water to the thermodynamics of binding IHF to H' DNA

To characterize driving forces and driven processes in formation of a large-interface, wrapped protein-DNA complex analogous to the nucleosome, we have investigated the thermodynamics of binding the 34-base pair (bp) H′ DNA sequence to the Escherichia coli DNA-remodeling protein integration host factor (IHF). Isothermal titration calorimetry and fluorescence resonance energy transfer are applied to determine effects of salt concentration [KCl, KF, K glutamate (KGlu)] and of the excluded solute glycine betaine (GB) on the binding thermodynamics at 20 °C. Both the binding constant K_obs and enthalpy ΔH°_obs depend strongly on [salt] and anion identity. Formation of the wrapped complex is enthalpy driven, especially at low [salt] (e.g., ΔH^o_obs = − 20.2 kcal·mol^− 1 in 0.04 M KCl). ΔH°_obs increases linearly with [salt] with a slope (dΔH°_obs/d[salt]), which is much larger in KCl (38 ± 3 kcal·mol^− 1 M^− 1) than in KF or KGlu (11 ± 2 kcal·mol^− 1 M^− 1). At 0.33 M [salt], K_obs is approximately 30-fold larger in KGlu or KF than in KCl, and the [salt] derivative SK_obs = dlnK_obs/dln[salt] is almost twice as large in magnitude in KCl (− 8.8 ± 0.7) as in KF or KGlu (− 4.7 ± 0.6).A novel analysis of the large effects of anion identity on K_obs, SK_obs and on ΔH°_obs dissects coulombic, Hofmeister, and osmotic contributions to these quantities. This analysis attributes anion-specific differences in K_obs, SK_obs, and ΔH°_obs to (i) displacement of a large number of water molecules of hydration [estimated to be 1.0(± 0.2) × 10³] from the 5340 Å² of IHF and H′ DNA surface buried in complex formation, and (ii) significant local exclusion of F⁻ and Glu⁻ from this hydration water, relative to the situation with Cl⁻, which we propose is randomly distributed. To quantify net water release from anionic surface (22% of the surface buried in complexation, mostly from DNA phosphates), we determined the stabilizing effect of GB on K_obs: dlnK_obs/d[GB]  = 2.7 ± 0.4 at constant KCl activity, indicating the net release of ca. 150 H₂O molecules from anionic surface.     

Analysis of multitemperature isothermal titration calorimetry data at very low c: Global beats van't Hoff

Isothermal titration calorimetry data for very low c (≡K[M]₀) must normally be analyzed with the stoichiometry parameter n fixed — at its known value or at any reasonable value if the system is not well characterized. In the latter case, ΔH° (and hence n) can be estimated from the T-dependence of the binding constant K, using the van't Hoff (vH) relation. An alternative is global or simultaneous fitting of data at multiple temperatures. In this Note, global analysis of low-c data at two temperatures is shown to estimate ΔH° and n with double the precision of the vH method.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Preparation and electrochemical properties of novel cyclic dinuclear acetylacetonato ruthenium complexes doubly bridged with sulfur and/or disulfur

A novel cyclic dinuclear acetylacetonato ruthenium complex doubly bridged with sulfur and/or disulfur at the γ-position of acetylacetonato ligand has been obtained by two different synthetic methods. The molecular structure of the dinuclear complex has been determined by single crystal X-ray diffraction study. Other two cyclic dinuclear β-diketonato ruthenium complexes were also prepared in good yields by the reaction of single bridged dinuclear complexes as starting materials with disulfur dichloride. The cyclic voltammograms of all the dinuclear complexes exhibit two one-electron reduction and oxidation waves in acetonitrile (AN) and dichloromethane (DM). The comproportionation constants (K_c) for mixed-valence state of both Ru^II/Ru^III and Ru^III/Ru^IV were evaluated in both solvents at 25 or −30 °C. The values of both K_c (Ru^II/Ru^III) and log₁₀ K_c (Ru^III/Ru^IV) for double bridged complex are large compared to those of corresponding single bridged complexes. This fact was rationally explained by the double bridging effect caused by the spread of electronic communication and also demonstrated the usefulness of the double bridged dinuclear complexes.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.