Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.     

Stability and networks of hydrogen bonds of the collagen triple helical structure: influence of pH and chaotropic nature of three anions.

The thermal stability of the trimeric species formed by seven type I collagen CNBr peptides was determined at neutral and acidic pH. Melting temperature of peptide trimers and free energy change for monomer to trimer transition were used as indices of trimer stability. A greater stability at neutral pH than at acidic pH was found for all peptides analysed because in most conditions an entropic gain overwhelms an enthalpic cost. Enthalpic reasons are prevailing only in some conditions of the more acidic peptides. The overlap zone of type I collagen fibrils is more basic than the gap zone and is therefore more sensitive to variations of pH from neutral to acidic, e.g. in bone degradation when osteoclasts acidify the lacuna lying between cell and bone. Peptide trimer stability in neutral conditions is influenced also by the chaotropic nature and the concentration of three anions: chloride, sulfate and phosphate. This was more evident for sulfate at the highest concentration used (0.5 M) when a greater stability is caused by entropic reasons. The contribution of hydroxyproline to the stability of peptide trimers is greater at neutral than at acidic pH, particularly at the highest concentration of sulfate. All our data indicate that pH, chaotropic nature and concentration of three anions influence the networks of hydrogen bonds present in the collagen triple helical structure.     

Stability studies of chondroitin sulfate.

The stability of chondroitin sulfate (CS) was studied under acidic, neutral and basic conditions at 30 and 60 degrees C. CS is remarkably stable under neutral conditions at low temperature, while it degrades at 60 degrees C producing low-molecular-mass fragments and desulfated products. This decomposition process begins at ca. 500-600 h and is consistent with an acid-catalyzed hydrolysis of glycosidic linkages caused by a drop in pH resulting from acidic products. Under basic conditions, a breakdown of glycosidic linkages causes a decrease in molecular mass due to the beta-elimination reaction, confirmed by a strong increase of absorbance at 232 nm and 1H NMR. Virtually no loss of O-sulfate groups can be detected in the base-treated CS. Under acidic conditions, the molecular mass decreases probably through hydrolysis of polysaccharidic linkages resulting in an increased number of reducing end groups. Little or no beta-elimination occurs. A loss of O-sulfate groups was detected, producing desulfated derivatives.     

Crystal structures of native and xylosaccharide-bound alkali thermostable xylanase from an alkalophilic Bacillus sp. NG-27: structural insights into alkalophilicity and implications for adaptation to polyextreme conditions

Crystal structures are known for several glycosyl hydrolase family 10 (GH10) xylanases. However, none of them is from an alkalophilic organism that can grow in alkaline conditions. We have determined the crystal structures at 2.2 Angstroms of a GH10 extracellular endoxylanase (BSX) from an alkalophilic Bacillus sp. NG-27, for the native and the complex enzyme with xylosaccharides. The industrially important enzyme is optimally active and stable at 343 K and at a pH of 8.4. Comparison of the structure of BSX with those of other thermostable GH10 xylanases optimally active at acidic or close to neutral pH showed that the solvent-exposed acidic amino acids, Asp and Glu, are markedly enhanced in BSX, while solvent-exposed Asn was noticeably depleted. The BSX crystal structure when compared with putative three-dimensional homology models of other extracellular alkalophilic GH10 xylanases from alkalophilic organisms suggests that a protein surface rich in acidic residues may be an important feature common to these alkali thermostable enzymes. A comparison of the surface features of BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in the polyextremophilicity of proteins, where understanding the structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. The work brings to the fore the role of the nature and composition of solvent-exposed residues in the adaptation of enzymes to polyextreme conditions, as in BSX.     

Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3-8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.     

Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH.

The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.     

Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions

Chitosan is a natural biopolymer whose rich amine functionality confers water solubility at low pH. At higher pH's (greater than 6. 5), the amines are deprotonated and chitosan is insoluble. To attain water solubility under basic conditions we enzymatically grafted the hydrophilic compound chlorogenic acid onto chitosan. Despite its name, chlorogenic acid is a nonchlorinated phenolic natural product that has carboxylic acid and hydroxyl functionality. The enzyme in this study was tyrosinase, which converts a wide range of phenolic substrates into electrophilic o-quinones. The o-quinones are freely diffusible and can undergo reaction with the nucleophilic amino groups of chitosan. Using slightly acidic conditions (pH = 6), it was possible to modify chitosan under homogeneous conditions. When the amount of chlorogenic acid used in the modification reaction exceeded 30% relative to chitosan's amino groups, the modified chitosan was observed to be soluble under both acidic and basic conditions, and to have a pH window of insolubility at near neutral pH. 1H NMR spectra confirmed that chitosan was chemically modified, although the degree of modification was low. Copyright 1999 John Wiley & Sons, Inc.     

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Insights from molecular dynamics simulations into pH-dependent enantioselective hydrolysis of ibuprofen esters by Candida rugosa lipase

An interesting observation was found during our continued studies on the hydrolysis of ibuprofen esters by Candida rugosa lipase (CRL). An important role is played by pH in the stereospecific hydrolysis of these esters. The flap region of CRL plays a significant role in the access of the substrate to the active site of the enzyme. At pH 5.6, 48% of the methyl ester and 5% of the butyl ester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2, 9% of methyl ester and 45% of the butyl ester of ibuprofen was hydrolysed in a identical reaction time using CRL. This lead us to assume that CRL prefers the methyl ester of ibuprofen as a substrate at an acidic pH and the butyl ester of ibuprofen at a neutral pH. Therefore, in order to understand the role of pH in the substrate selection by CRL for the esters of ibuprofen we used the crystallographic coordinates of the open form of the CRL (1CRL) for molecular dynamics (MD) simulations under acidic and neutral conditions for 2 ns using GROMACS. The final structures obtained after simulation in acidic and neutral conditions were compared with the energy-minimized structure, and the root-mean-square deviations (r.m.s.ds) were calculated. The r.m.s.d. of the CRL flap at neutral pH was found to be greater than that of the CRL flap at acidic pH. The extent to which the flap opens at neutral pH allowed the bulkier substrate, the butyl ester of ibuprofen, to diffuse into the active site and provides the best enzyme-substrate fit for this specific substrate. At acidic pH there is a decreased opening of the flap thereby accommodating a more compact substrate, namely the methyl ester of ibuprofen. Thus, simulation experiments using MD provide reasonable insight for the pH-dependent substrate selectivity of CRL in aqueous environments.     

Self-association and stability of the ApoE isoforms at low pH: implications for ApoE-lipid interactions

Apolipoprotein E (apoE) isoforms are known to differentially accumulate in the lysosomes of neuronal cells, and the deleterious effects of the apoE4 isoform in Alzheimer's disease may relate to its properties at the low lysosomal pH. However, the effect of pH on the molecular properties of full-length apoE is unclear. Here we examine the pH dependence of the monomer-dimer-tetramer reaction, of lipid binding, and of the stability of the three major apoE isoforms. Using FRET measurements, we find that the association-dissociation behavior of apoE proteins changes dramatically with changes in pH. At pH 4.5, approximating the pH of the lysosome, rate constants for association and dissociation are 2-10 times faster than those at pH 7.4. Aggregation beyond the tetrameric form is also more evident at lower pH values. Stability, as measured by urea denaturation at pH 4.5, is found to be considerably greater than that at neutral pH and to be isoform dependent. Lipid binding, as measured by turbidity clearance of unilamellar vesicles of DMPC, is faster at acidic pH values and consistent with our previous hypothesis that it is only the monomeric form of apoE that binds lipid tightly. Since apoE is more stable at pH 4.5 than at neutral pH, the more rapid apoE-lipid interactions at low pH are not correlated with the stability of the apoE isoforms, but rather to the faster association-dissociation behavior. Our results indicate that pathological behavior of apoE4 may arise from altered molecular properties of this protein at the acidic pH of the lysosome.     

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F (p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% (p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% (p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.     

Toxic impact of phosphamidon on protein degradation in phasic and tonic muscles of marine prawn, Penaeus indicus (H. Milne Edwards).

Levels of various protein fractions, (sarcoplasmic, myosin, actin, non-collagen and collagen) and the rate of their degradation by proteases were studied in phasic and tonic muscles of marine prawn, Penaeus indicus following acute (2 d) and chronic (15 d) exposure to sublethal concentration of phosphamidon. During exposure, greater decrease in sarcoplasmic protein fraction was observed in phasic muscle as compared to other myofibrillar proteins. But the sarcoplasmic protein content showed an elevation in tonic muscle. The changes in protein fractions were more pronounced during acute exposure than chronic exposure both in phasic and tonic muscles. These changes were correlated with the elevation of the acidic, neutral and basic protease activities during acute and chronic exposure. Free amino acids were increased during acute exposure, while they showed a significant decrease during chronic exposure in both the muscles. These results indicate that protein metabolism in both phasic and tonic muscles was significantly altered following phosphamidon exposure. These differential responses observed at acute and chronic exposure indicate the operation of compensatory mechanisms to mitigate the phosphamidon toxic stress.     

Molecular dynamics simulation of the unfolding of the human prion protein domain under low pH and high temperature conditions

Four 10-ns molecular dynamics (MD) simulations of the human prion protein domain (HuPrP 125-228) in explicit water solution have been performed. Each of the simulations mimicked a different environment of the protein: the neutral pH environment was simulated with all histidine residues neutral and bearing a ND proton and with other titratable side chains charged, the weakly acidic environment was simulated with all titratable side chains charged, the strongly acidic environment was simulated with all titratable side chains protonated. The protein in neutral pH environment was simulated at both ambient (298 K) and higher (350 K) temperatures. The native fold is stable in the neutral pH/ambient temperature simulation. Through out all other simulations, a quite stable core consisted of 10-20 residues around the disulfide bond retain their initial conformations. However, the secondary structures of the protein show changes of various degrees compared to the native fold, parts of the helices unfolded and the beta-sheets extended. Our simulations indicated that the heat-induced unfolding and acid-induced unfolding of HuPrP might follow different pathways: the initial stage of the acid-induced unfolding may include not only changes in secondary structures, but also changes in the tertiary structures. Under the strongly acidic condition, obvious tertiary structure changes take place after 10-ns simulation, the secondary structure elements and the loops becoming more parallel to each other, resulting in a compact state, which was stabilized by a large number of new, non-native side chain-side chain contacts. Such tertiary structure changes were not observed in the higher temperature simulation, and intuitively, they may favor the further extension of the beta-sheets and eventually the agglomeration of multiple protein molecules. The driving forces for this tertiary structure changes are discussed. Two additional 10-ns MD simulations, one with Asp202 protonated and the other with Glu196 protonated compared to the neutral pH simulation, were carried out. The results showed that the stability of the native fold is very subtle and can be strongly disturbed by eliminating a single negative charge at one of such key sites. Correlations of our results with previous experimental and theoretical studies are discussed.     

Drug-induced zeta potential changes in liposomes studied by laser Doppler spectroscopy

The technique of laser Doppler spectroscopy is used to measure the electrophoretic mobility of liposomes under the influence of one beta-blocking agent and three local anesthetics. All four drugs decrease the mobility (i.e., the zeta potential) of negatively charged phospholipids (soybean lipids, phosphatidylserine and cardiolipin). The mobility of electrostatically neutral pure phosphatidylcholine (zero mobility under control conditions at pH 7 and 4) is increased linearly with the logarithm of drug concentration, indicating binding and incorporation of positively charged drug molecules. The sequence of strength of activity, measured by zeta-potential changes, corresponds to that found in biological tissues: propranolol greater than tetracaine greater than lidocaine greater than procaine. For purely negatively charged lipids (phosphatidylserine, cardiolipin) the activity of the drug is higher at acidic pH, (pH 4), while for electrostatically neutral (phosphatidylcholine) or partly neutral (soybean) lipid liposomes drug activity is about the same at pH 9, 7 and 4. A Hill plot of the data reveals noncooperative drug binding. From the line width of the scattering power spectrum the mean particle radius and the average interparticle distance in the samples are determined.     

Role of Neurotoxin Associated Proteins in the Low pH Induced Structural Changes in the Botulinum Neurotoxin Complex

Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of ?1.8 × 10⁵° cm²/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the T_m for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.     

Poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers that respond sharply to temperature and pH

Temperature- and pH-sensitive random copolymers of N-isopropylacrylamide (NIPAAm) and propylacrylic acid (PAA) were prepared using the reversible addition fragmentation chain transfer (RAFT) polymerization method. The lower critical solution temperatures (LCSTs) (or phase separation temperatures) of the NIPAAm-co-PAA copolymer solutions were measured by the cloud-point method. At slightly acidic conditions, the LCST decreased with increase in PAA content, which suggests that the hydrophobic propyl group of PAA has a greater influence on the LCST than the polar carboxylic acid group at those conditions. An increase of pH led to a significant increase in LCST of the copolymers due to the ionization of the -COOH group. The LCSTs were studied as a function of copolymer composition over the pH range from 5.0 to 7.0. Because the pK(a) of the polymers can be tuned to fall close to neutral pH, these polymer compositions can be designed to have phase transitions triggered near physiological pH or at slightly acidic pH values that fall within acidic gradients found in biology. The NIPAAm-co-PAA copolymers thus display tunable properties that could make them useful in a variety of molecular switching and drug delivery applications where responses to small pH changes are relevant.     

Toxicity of 2,3,5,6-tetrachlorophenol to willow trees (Salix viminalis)

Chlorinated phenols have been intensively investigated from an eco-toxicological point of view, however almost nothing is known about toxicity of tetrachlorophenol (TeCP) to higher terrestrial plants. This article applied the willow tree acute toxicity test to study the toxicity of 2,3,5,6-TeCP to willows Salix viminalis (S. viminalis) at neutral and acidic conditions (roughly pH 7 and 4) with inhibition of transpiration as toxic endpoint. At neutral pH the EC₅₀ was >10 mg L^?1 while the EC₅₀ at acidic conditions was 0.32 ± 0.17 mg L^?1, clearly indicating that toxicity is exerted by the non-ionic chemical fraction. Standard tests running at neutral pH are therefore not capturing the full toxicity of weak acids and bases.     

Temperature-induced structural changes of apo-lactoferrin and their functional implications: a molecular dynamics simulation study

Lactoferrin (Lf) is an iron-binding glycoprotein present in secretory fluids from human and bovine sources. Sequence alignment was employed to identify a region on the C-lobe of Lf capable of binding to bacterial cell surfaces, followed by all-atom explicit solvent molecular dynamics simulations to study the conformational changes of Lf after exposure to three processing temperatures: pasteurisation (72°C), spray drying (90°C) and ultra-high temperature (UHT) (127°C). Below 90°C, the simulations indicate relatively minor changes in overall protein structure. At UHT conditions (127°C), however, marked disruptions to protein structure were found as demonstrated by a substantial decrease in protein dimensions due to collapse in the inter-lobe region. There was also a marked increase in residue fluctuations in several regions of known functional importance, including antibacterial, iron-binding, and putative membrane binding regions, the latter of which is stabilised by a triplet of hydrophobic residues comprised of Leu446, Trp448 and Leu451 at low temperature, but which are disrupted under UHT conditions. A unique network analysis confirmed these results as demonstrated by large clusters of residues with increased dynamical correlation in the N-terminal lobe.     

Adsorption of 6-mercaptopurine and 6-mercaptopurine-ribosideon silver colloid: a pH-dependent surface-enhanced Raman spectroscopy and density functional theory study. II. 6-mercaptopurine-riboside

Surface-enhanced Raman spectroscopy (SERS) has been applied to characterize the interaction of 6-mercaptopurine-ribose (6MPR), an active drug used in chemotherapy of acute lymphoblastic leukemia, with a model biological substrate at therapeutic concentrations and as function of the pH value. Therefore, a detailed vibrational analysis of crystalline and solvated (6MPR) based on Density Functional Theory (DFT) calculations of the thion and thiol tautomers has been performed. 6MPR adopts the thion tautomeric form in the polycrystalline state. The SERS spectra of 6MPR and 6-mercaptopurine (6MP) recorded on silver colloid provided evidence that the ribose derivative shows different adsorption behavior compared with the free base. Under acidic conditions, the adsorption of 6MPR on the metal surface via the N7 and possibly S atoms was proposed to have a perpendicular orientation, while 6MP is probably adsorbed through the N9 and N3 atoms. Under basic conditions both molecules are adsorbed through the N1 and possibly S atoms, but 6MP has a more tilted orientation on the silver colloidal surface while 6MPR adopts a perpendicular orientation. The reorientation of the 6MPR molecule on the surface starts at pH 8 while in the case of 6MP the reorientation starts around pH 6. Under basic conditions, the presence of the anionic molecular species for both molecules is suggested. The deprotonation of 6MP is completed at pH 8 while the deprotonation of the riboside is finished at pH 10. For low drug concentrations under neutral conditions and for pH values 8 and 9, 6MPR interacts with the substrate through both N7 and N1 atoms, possibly forming two differently adsorbed species, while for 6MP only one species adsorbed via N1 was evidenced.