Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

Conformational change and inactivation of arginine kinase from shrimp Feneropenaeus chinensis in oxidized dithiothreitol solutions.

The effect of oxidized dithiothreitol (DTT) on the conformation and function of arginine kinase from shrimp Feneropenaeus chinensis was investigated with the methods of intrinsic fluorescence, ANS fluorescence, size exclusion chromatography (SEC), sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), and activity assay. The excess molecular oxidized dithiothreitol could result in a loss of activity and conformational change of arginine kinase. The oxidized arginine kinase was characterized by monitoring the changes of fluorescence emission wavelength (excitation wavelength: 295 nm) and the intensity of 1-anilino-8-naphthalenesulfonate (ANS) binding (excitation wavelength: 380 nm) to the protein. The results of fluorescence spectra showed that the presence of oxidized DTT could result in a marked change in the enzyme tertiary structure. The conformational changes of native and oxidized arginine kinase are induced by the presence of the full set of transition state analog (TSA) components. The results of size exclusion chromatography and SDS-PAGE indicated that no disulfide bond was formed among the protein molecules in the oxidized-DTT solution.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k_i), increasing the transition free energy changes (ΔΔG_i) and increasing the value for the midpoint of denaturation (C_m). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.     

Proteolytic susceptibility of creatine kinase isozymes and arginine kinase

The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.     

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

Aggregation and folding of recombinant human creatine kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.     

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu³¹⁴ decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe³¹⁵ mutants including the AAloop which retained Glu³¹⁴ had modest AK activity (5–84% of the wild type). All the results above suggested that Glu³¹⁴ played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu³¹⁴ mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe³¹⁵ in dimeric AK has a different role from His³¹⁵ in monomeric AK. This might contribute to the stabilization of the native conformation, while His³¹⁵ in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.     

Interaction of metal nanoparticles with recombinant arginine kinase from Trypanosoma brucei: Thermodynamic and spectrofluorimetric evaluation

Background

Trypanosoma brucei, responsible for African sleeping sickness, is a lethal parasite against which there is need for new drug protocols. It is therefore relevant to attack possible biomedical targets with specific preparations and since arginine kinase does not occur in humans but is present in the parasite it becomes a suitable target.

Methods

Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles.

Results

The enzyme only had one binding site. At 25 °C the dissociation (K_d) and Stern–Volmer constants (K_SV) were 15.2 nM, 0.058 nM^− 1 [Ag]; and 43.5 nM, 0.052 nM^− 1 [Au] and these decreased to 11.2 nM, 0.041 nM^− 1 [Ag]; and 24.2 nM, 0.039 nM^− 1 [Au] at 30 °C illustrating static quenching and the formation of a non-fluorescent fluorophore–nanoparticle complex. Silver nanoparticles bound to arginine kinase with greater affinity, enhanced fluorescence quenching and easier access to tryptophan molecules than gold. Negative ΔH and ΔG values implied that the interaction of both Ag and Au nanoparticles with arginine kinase was spontaneous with electrostatic forces. FRET confirmed that the nanoparticles were bound 2.11 nm [Ag] and 2.26 nm [Au] from a single surface tryptophan residue.

Conclusions

The nanoparticles bind close to the arginine substrate through a cysteine residue that controls the electrophilic and nucleophilic characters of the substrate arginine–guanidinium group crucial for enzymatic phosphoryl transfer between ADP and ATP.

General significance

The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.     

Effects of osmolytes on arginine kinase from Euphausia superba: A study on thermal denaturation and aggregation

Investigations of energy-related enzymatic properties may provide valuable information about the mechanisms that are involved in the adaptation to extreme climatic environments. The protective effects of osmolytes on the thermal denaturation and aggregation of arginine kinase from E. superba (ESAK) was investigated. When the concentration of glycine, proline and glycerol increased, the relative activation was significantly enhanced, while the aggregation of ESAK during thermal denaturation was decreased. Spectrofluorometry results showed that the presence of these three osmolytes significantly decreased the tertiary structural changes of ESAK and that thermal denaturation directly induced ESAK aggregation. The results demonstrated that glycine, proline and glycerol not only prevented ESAK from inactivation and unfolding but also inhibited aggregation by stabilizing the ESAK conformation. We measured the ORF gene sequence of ESAK by RACE, and built the 3D structure of ESAK and osmolytes by homology models. The results showed that the docking energy was relatively low and that the clustering groups were spread to the surface of ESAK, indicating that osmolytes directly protect the surface of the protein. Our study provides important insight into the protective effects of osmolytes on ESAK folding.     

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.     

Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.     

Impact of inter-subunit interactions on the dimeric arginine kinase activity and structural stability

Arginine kinase (AK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphoarginine to ADP in invertebrates. In this study, the inter-subunit hydrogen bonds between the Q53 and D200 and between D57 and D200 were disrupted to explore their roles in the activity and structural stability of Stichopus japonicus (S. japonicus) AK. Mutating Q53 and/or D57 to alanine (A) can cause pronounced loss of activity and substrate synergism, and cause distinct conformational changes. Spectroscopic experiments indicated that mutations destroying the inter-subunit hydrogen bonds impaired the structure of dimer AK, and resulted in a partially unfolded state. The inability to fold to the functional compact state made the mutants prone to be inactivated and aggregate under environmental stresses. Restoring hydrogen bonds in Q53E and D57E mutants could rescue the loss of activity and substrate synergism, and conformational changes. All those results suggested that the inter-subunit interactions played a key role in keeping the activity, substrate synergism and structural stability of dimer AK. The result herein may provide a clue in understanding the folding and self-assembly processes of oligomeric proteins.     

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L^− 1 of LB medium. Some parameters for the renatured and soluble forms of AK, including K_m, K_d, specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest k_cat and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.     

Studies of the unfolding of an unstable mutant of staphylococcal nuclease: Evidence for low temperature unfolding and compactness of the high temperature unfolded state

Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, ΔC_p, between the unfolded and native states. This analysis gives a ΔC_p of 2.2 kcal/mol/·K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These ΔC_p values are consistent with significant population of the cold unfolded state at ∼0°C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0°C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Proteins 28:227–240, 1997 © 1997 Wiley-Liss Inc.     

Estimation of pH effect on the structure and stability of kinase domain of human integrin-linked kinase

Integrin-linked kinase (ILK) is an evolutionarily conserved Ser/Thr protein kinase, involved in many physiological functions such as signal transduction, actin rearrangement, cell proliferation, migration, polarisation, angiogenesis and apoptosis. An increased expression of ILK is associated with different cancers and thus considered as an attractive target for cancer therapy. We have successfully cloned, expressed and purified the kinase domain (193–446 residues) of ILK. To see the effect of pH on the structure and conformation, we performed circular diachroism, fluorescence and absorbance measurements in a wide range of pH conditions. We observed that within the range of pH 7.5–11.0, ILK_193–446 maintains its both secondary and tertiary structures. While visible aggregates were observed under the acidic pH 2.0–5.5 conditions, in order to complement these observations, we have performed molecular dynamics simulations of this kinase domain by mimicking diverse pH conditions which enabled us to see conformational preferences of the protein under such conditions. A significant correlation between the spectroscopic and molecular dynamics simulation was observed. These findings are useful to understand the conformation of ILK protein under certain pH condition which may be further implicated in the drug design and discovery.     

Role of arginine kinase in Paramecium tetraurelia (Ciliophora,Peniculida): Subcellular localization of AK3 and phosphoarginine shuttle system in cilia

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia.     

Utility of arginine kinase for resolution of phylogenetic relationships among Brachyuran genera and families

The molecular phylogenetics of decapod crustaceans has been based on sequence data from a limited number of genes. These have included rapidly evolving mitochondrial genes, which are most appropriate for studies of closely related species, and slowly evolving nuclear ribosomal RNA genes, which have been most useful for resolution of deep branches within the Decapoda. Here we examine the utility of the nuclear gene that encodes arginine kinase for phylogenetic reconstruction at intermediate levels (relationships among genera and families) within the decapod infraorder Brachyura (the true crabs). Analyses based on arginine kinase sequences were compared and combined with those for the mitochondrial cytochrome oxidase I gene. All of the genera in our taxon sample were resolved with high support with arginine kinase data alone. However, some of these genera were grouped into clades that are in conflict with recognized brachyuran families. A phylogeny based on cytochrome oxidase I was consistent with the arginine kinase phylogeny, but with weaker support. A recently proposed measure of phylogenetic informativeness indicated that arginine kinase was generally more informative than cytochrome oxidase I for relationships above the level of genus. Combined analysis of data from both genes provided strong support for clades that are in conflict with current assignments of genera to the families Epialtidae, Mithracidae, Pisidae, and Portunidae.     

Expression,purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mgL(-1) of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.     

Functional expression of arginine kinase improves recovery from pH stress of Escherichia coli

Acid stress in Escherichia coli involves a complex resource- and energy-consuming response mechanism. By overexpression of arginine kinase from Limulus polyphemus in E. coli, we improved the recovery from a transient pH stress. While wild type E. coli resumed growth after a transient pH reduction to pH 3 for 1 h with a rate that was 25% lower than before the stress, the arginine kinase expressing strain continued to grow as rapidly as before. This effect is presumably caused by the physiological function of arginine kinase as a short term energy buffer in the form of phosphoarginine, but a pH-buffering effect cannot be excluded.