The Energy and Nutritional Value of Meat of Broiler Chickens Fed with Various Addition of Wheat Germ Expeller.

The study concerns the effect of wheat germ expeller (WGE) as a feed additive given to male Ross-308 broiler chickens on their meat's energy and nutritional value, and coverage of nutrient reference values-requirements (NRV-R) of consumers for particular minerals. The chickens in the control group (CT-Control Treatment) were fed a standard complete mix. The experimental groups (EX5, EX10, EX15) were given a feed in which wheat middling was replaced with 5, 10, and 15% WGE. The breast and thigh muscles of 32 randomly selected chickens (8 in each group) were analyzed. More water, crude protein, P, Mg, Fe, Cu, and Mn were determined in the breast muscles, and more crude fat, crude ash, Ca, and Zn in the thigh muscles. Chickens from the CT group consumed significantly (p ≤ 0.01) less feed per body weight than those from groups EX5 to EX15, but achieved the highest body weight per 100 g of consumed feed. A higher (p ≤ 0.01) feed, energy, crude protein, and crude fat intake was observed in groups EX5 to EX15 compared to CT. The higher (p ≤ 0.01) value of protein efficiency ratios was indicated in the CT group. The WGE additive did not impact the muscles' energy values but affected the nutritional value. The daily consumption of 100 g of breast muscles to a large extent covers the consumer NRV-R for P, Mg Fe, Cu, and Mn. However, thigh muscles cover the NRV-R to a greater extent for Ca and Zn. The EX15, EX5, and EX10 muscles covered most of the NRV-R for P, Ca, and Mg, while the CT muscles did the same for Zn and Mn. Adding 5% WGE to broiler feed is optimal as it does not impair the nutritional value of the muscles.

Technological properties, chemical composition, texture profile, and sensory evaluation of goose muscles from Polish native breeds.

The study aimed to determine the technological and sensory properties of the breast and thigh muscles of geese from the Polish native varieties: Kartuska (Ka) and Suwalska (Su) (from northern Poland) as well as Lubelska (Lu) and Kielecka (Ki) (from southern Poland). The color parameters: L*, a*, b*, ΔE, C, h°, total heme pigments (THPs), and share of myoglobin (Mb), metmyoglobin (MMb), and oxymyoglobin (MbO2) in muscles were determined. In terms of technological properties, the following were determined: pH24, water-binding capacity (WBC), water-holding capacity (WHC), cooking (CL), and roasting losses (RL). In addition, a sensory evaluation of the raw meat color was performed. In roasted meat, a sensory evaluation and texture profile analysis (TPA) were carried out, as well as the shear force (SF) and chemical composition were determined. Roasted muscles of varieties native to northern Poland (Ka and Su) were higher in lipids (P≤0.05) than the muscles of southern varieties (Lu and Ki). Ka meat had the highest protein content, and Lu meat had the lowest (P≤0.05). The raw muscle color sensory evaluation results, the THP, and the L* and ΔE values indicated that the darkest color among the studied genotypes were the Ka muscles, and the lightest was Ki meat (P≤0.05). Lu's muscles are distinguished by better usability for processing and culinary purposes than the muscles of the other genotypes due to high pH24, WBC, WHC, and low RL and CL of thigh muscles, as well as high WHC and low RLs of the breast muscles (P≤0.05). Due to the tenderness, juiciness, and high general evaluation (P≤0.05), the best sensory features among the studied genotypes were found in the Ka breast and thigh muscles. The low SF value proved the higher tenderness of Ka geese muscles.

The Effects of Sous Vide, Microwave Cooking, and Stewing on Some Quality Criteria of Goose Meat.

Background: Heat treatment methods including frying (with and without fat or oil), deep frying, oven roasting, grilling, charcoal roasting, broiling, steaming, and microwave cooking promote a cascade of adverse changes in the functional properties of meat, including protein fraction, lipid oxidation, and loss of some vitamins and mineral compounds. The aim of this study was to evaluate the influence of three cooking methods (sous vide (SV), microwave (M) cooking, and stewing (S)) on the basic chemical composition, cholesterol content, energy value, mineral concentration, and retention coefficients in goose meat. Methods: Basic chemical composition and mineral analysis were determined using AOAC methods. Total cholesterol content was established using the HPLC method. Results: Both types of goose meat (without and with skin) and heat treatment had a significant effect on nutrient values, mineral concentration, and retention coefficients. The S meat was characterized by a higher protein content than M and SV meat, and had the lowest fat, protein, and cholesterol retention, among other methods. The M meat had lower total cholesterol content than SV and S meat. There were significant differences in energy value for SV, M, and S meats. The SV meat contained less P, Mg, Fe, Zn, and more Na and K than the M and S samples. The highest values of Zn, Mg, and Fe content and the lowest of K and Ca were recorded in S meat compared with the SV and M samples. The retention coefficients of P, Mg, Na, Ca, and K in S meat were lower than in the SV and M samples. The meat without skin was characterized by a lower energy value, fat content, retention of proteins, and cholesterol, but higher fat retention than skin samples. This meat contained more minerals such as P, Mg, Fe, K, Na, and less Ca than skin meat. Higher retention coefficients were observed for Zn, P, Mg, Ca, and lower were observed for Na, Fe, and K in meat without skin than in samples with skin. Conclusions: From a dietary point of view, the most beneficial were SV muscles without skin. Whereas, taking into account the protein, fat content, and retention coefficients of fat, cholesterol, Zn, and Na, the most optimal form of cooking for meat with skin seems to be stewing. These results may be used by consumers in making dietary choices by taking into account the type of goose meat and kind of heat treatment.

Impact of various types of heat processing on the energy and nutritional values of goose breast meat.

The purpose of this study was to examine the impact of various types of heat processing used by consumers (water bath cooking WBC, oven convection roasting OCR, grilling G, pan frying PF) on the energy and the nutritional value of goose breast meat (with and without skin). The material used in the study comprised 72 breast muscles cut from carcasses of 17-wk-old White Koluda geese. The energy value (MJ), the chemical composition (water, fat, protein, ash) and mineral composition (phosphorus P, sodium Na, calcium Ca, potassium K, magnesium Mg, iron Fe, zinc Zn, cooper Cu, manganese Mn) were determined in both raw and thermally processed muscles. It has been concluded that various methods of heat processing have a significant impact on the energy and nutritional values of meat. From a dietary point of view, the most beneficial was OCR meat without skin, and WBC, OCR, PF meat with skin as well, since it had the lowest energy value as well as content and retention of fat, phosphorus, and sodium. However, as for the content of the other minerals and their retention, WBC seems to be the optimal form of heat treatment of skinless muscles. 100 g of such meat provides 3.1; 33.7; 145; 180 and 9% Nutrient Reference Values-Requirements (NRVs-R) for Ca, Mg, Fe, Cu, and Mn respectively in a diet of an adult person. As for meat with skin, the optimal method of heat processing to retain minerals is grilling. 100 g of meat processed in this way provides 3.9; 39.7; 125.7; 175; 6 and 12.7% NRVs-R of Ca, Mg, Fe, Cu, and Mn. It follows from the above information that goose breast meat, as analyzed here, cannot be considered as a source of calcium since it provides less than 4% of NRVs-R. The results of the study will be useful for the consumers' nutritional choices. The geese breast meat, depending on the heat processing used and the content of skin, may be a valuable component of a varied diet, providing nutrients and minerals.

The protein and fat quality of thigh muscles from Polish goose varieties.

This study aimed to evaluate the nutritional value of thigh meat from 4 Polish geese varieties. Protein, fat, and cholesterol content, as well as amino acid and fatty acid profiles, were determined. Based on the percentage of amino acid in protein and fatty acids in meat lipids, the health lipid indices were calculated. The experimental material covered thigh muscles from 17-week-old Kartuska (Ka), Suwalska (Su), Lubelska (Lu), and Kielecka (Ki) geese reared in a semi-intensive system. Muscle protein content did not differ significantly between varieties. The protein content of the Ka, Su, Lu, and Ki goose meat was deemed high-value as it contained all the essential amino acids in the proportions consistent with standard protein values. The muscles of all the researched geese varieties were characterized by a high level of Lys, which indicates that this meat is a good source of it (AASLys 240-280%). Current findings showed that polyunsaturated fatty acids (PUFA)/saturated fatty acids (SFA) and PUFA n-6/n-3 ratios in Ka, Su, Lu, and Ki muscles were found to be within the optimum values for human diets. No significant differences were observed in monounsaturated fatty acids, PUFA, and unsaturated fatty acids (UFA) between the analyzed muscles. The meat of Ka and Su contained significantly more lipids than Lu and Ki. A more beneficial amino acid profile was found in Ka meat due to a higher content of PUFA n-3 and the best n-6/n-3 ratio in comparison with other varieties. The muscles of the Ka variety also contained the least cholesterol. However, the Ki goose muscles stood out among other varieties with the least percentage of SFA, the highest share of docosahexaenoic acid (C 22:6 n-3), as well as the most beneficial value of the following indices: UFA/SFA, hypocholesterolemic fatty acid/hypercholesterolemic fatty acid ratio, and nutritive value index. The thigh muscles of Ka, Su, Lu, and Ki were characterized by an atherogenicity index that met the levels of recommended values (<1) in the diet of a human being, while the thrombogenicity index was slightly higher than the recommended value (<0.5).

Fatty acid profiles and health lipid indices in the breast muscles of local Polish goose varieties.

The aim of this study was to evaluate the fatty acid profile and health lipid indices of meat from 3 Polish local goose varieties (Romanian-RO, Pomeranian-PO, and Subcarpathian-SB) and the commercial cross White Koluda goose (W31). Birds were fed ad libitum with the same complete feeds until 17 wk of age. The geese (n = 72) with body weight close to the arithmetic mean in particular flock were fasted for 12 h and slaughtered in an experimental slaughterhouse (18 females in each flock). Carcasses were stored at 2 to 4°C for 24 h. The breast muscles (m. pectoralis major) were cut out from the left side of carcass, separately vacuum-packed, and stored at -80°C until analysis. Fatty acid profile of meat was determined by gas chromatography and health lipid indices were calculated. The W31 muscles had a higher percentage of C 18:0 and a lower of C 16:0 than those of RO, PO, and SB geese. The W31 muscles were characterized by a significantly higher proportion of monounsaturated fatty acids (46.5%) than remaining ones (43.28%-PO, 43.38%-SB, and 44.24%-RO). The lowest proportion of polyunsaturated fatty acids was established for W31 muscles (22.05%). The breast muscles of RO, SB, and PO had more favorable polyunsaturated n-6 and n-3 fatty acid (PUFA)/ saturated fatty acid (SFA) ratio (0.85, 0.82, 0.83, respectively) than W31 geese (0.72). The current findings showed that UFA/SFA, PUFA/SFA, and PUFA n-6/n-3 ratios in RO and SB muscles were within the optimum values for human diets. No significant differences were observed in the atherogenic, thrombogenic, and hypocholesterolemic/hypercholesterolemic indices between the analyzed muscles. Commercial W31 geese breast muscles showed a lower value (43.90%) of peroxidizability index (PI) compared to SB (52.88%), PO (53.93%), and RO (53.47%). However, the higher values of the PUFA/SFA and PI in the meat of SB, PO, and RO birds may indicate a higher prohealth value of their meat.

2-OMe-lysophosphatidylcholine analogues are GPR119 ligands and activate insulin secretion from ßTC-3 pancreatic cells: Evaluation of structure-dependent biological activity.

GPR119 receptor has been proposed as a metabolic regulator playing a pivotal role in the modulation of glucose homeostasis in type 2 diabetes. GPR119 was identified on pancreatic ß cells and its ligands have the ability to enhance glucose-stimulated insulin secretion (GSIS). Lysophosphatidylcholine (LPC) was shown to potentiate GSIS and our present studies indicate that 2-methoxy-lysophosphatidylcholine (2-OMe-LPC) analogues, unable to undergo 1→2 acyl migration, stimulate GSIS from murine ßTC-3 pancreatic cells even more efficiently. Moreover, biological assays in engineered Tango™ GPR119-bla U2OS cells were carried out to ascertain the agonist activity of 2-OMe-LPC at GPR119. 2-OMe-LPC possessing in sn-1 position the residues of myristic, palmitic, stearic and oleic acid were also evaluated as factors regulating [Ca2+]i mobilization and cAMP levels. Extension of these studies to R- and S-enantiomers of 14:0 2-OMe-LPC revealed that the overall impact on GSIS does not depend on chirality, however, the intracellular calcium mobilization data show that the R enantiomer is significantly more active than S one. Taking into account differences in chemical structure between various native LPCs and their 2-methoxy counterparts the possible binding mode of 2-OMe-LPC to the GPR119 receptor was determined using molecular modeling approach.

The chemical synthesis and preliminary biological studies of phosphodiester and phosphorothioate analogues of 2-methoxy-lysophosphatidylethanolamine.

The chemical synthesis of phosphorothioate/phosphodiester analogues of 2-methoxy-lysophosphatidylethanolamine has been described. For the preparation of phosphorothioate derivatives oxathiaphospholane approach has been employed. The phosphodiester compounds were prepared by OXONE® oxidation of corresponding phosphorothioates. Each lysophospholipid analogue was synthesized as a series of four compounds, bearing different fatty acid residues both saturated (14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cytotoxicity of newly synthesized 2-methoxy-lysophosphatidylethanolamine derivatives was evaluated with resazurin-based method in prostate cancer PC3 cell line. The highest reduction of cell viability was noted for LPE analogues containing myristoyl acyl chain.

The chemical synthesis and cytotoxicity of new sulfur analogues of 2-methoxy-lysophosphatidylcholine.

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1 → 2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.

Intrinsic acid-base properties of a hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT): neighboring effects and isomeric equilibria.

The intrinsic acid-base properties of the hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1∙G2∙G3∙C4∙C5∙T6)=(HNPP)5⁻] have been determined by ¹H NMR shift experiments. The pKa values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10% D2O, 47 °C, I=0.1 M, NaClO4). These results quantify the release of H⁺ from the two (N7)H⁺ (G∙G), the two (N3)H⁺ (C∙C), and the (N1)H⁺ (A) units, as well as from the two (N1)H (G∙G) and the (N3)H (T) sites. Based on measurements with 2'-deoxynucleosides at 25 °C and 47 °C, they were transferred to pKa values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three pKa clusters occur, that is those encompassing the pKa values of 2.44, 2.97, and 3.71 of G2(N7)H⁺, G3(N7)H⁺, and A1(N1)H⁺, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H5(HNPP)(±) , the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4%, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no "cost". The eight pKa values for H5(HNPP)(±) are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1∙G2∙G3∙C4∙C5∙U6), where U=uridine, were calculated. Finally, the effect of metal ions on the pKa values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range.

The chemical synthesis of metabolically stabilized 2-OMe-LPA analogues and preliminary studies of their inhibitory activity toward autotaxin.

The chemical synthesis of five new metabolically stabilized 2-OMe-LPA analogues (1a-e) possessing different fatty acid residues has been performed by phosphorylation of corresponding 1-O-acyl-2-OMe-glycerols which were prepared by multistep process from racemic glycidol. The now analogues were subjected to biological characterization as autotaxin inhibitors using the FRET-based, synthetic ATX substrate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA (1e) appeared to be the most potent, showing ATX inhibitory activity similar to that of unmodified 1-O-oleoyl-LPA. Parallel testing showed, that similar trend was also observed for corresponding 1-O-acyl-2-OMe-phosphorothioates (2a-e, synthesized as described by us previously). 1-O-oleoyl-2-OMe-LPA (1e) was found to be resistant toward alkaline phosphatase as opposed to unmodified 1-O-oleoyl-LPA.

DNAzyme as an efficient tool to modulate invasiveness of human carcinoma cells.

In this study we evaluated efficiency of DNAzymes to modulate motility of cancer cells, an important factor in the progression and metastasis of cancers. For this purpose we targeted ß1 integrins that are predominant adhesive receptors in various carcinoma cell lines (CX1.1, HT29, LOVO, LS180, PC-3). To evaluate invasiveness of cancer cells, we used a transwell migration assay that allowed analyzing chemotactic migration of colon carcinoma cell lines across an ECM-coated membrane. Their adhesive properties were also characterized by the analysis of adhesion to fibronectin, laminin and collagen. In addition, the expression of major integrin subunits, selected intact ß1 integrins, and other adhesive receptors (ICAM, E-selectin, uPAR) was analyzed by flow cytometry. Inhibition of ß1 integrin expression by DNAzyme to ß1 mRNA almost abolished the invasiveness of the CX1.1, HT29, LS180, LOVO and PC-3 cells in vitro. These data show that DNAzymes to ß1 integrin subunit can be used to inhibit invasiveness of carcinoma cells.

The synthesis of di- and oligo-nucleotides containing a phosphorodithioate internucleotide linkage with one of the sulfur atoms in a 5'-bridging position.

A new type of internucleotide phosphorodithioate linkage is described, wherein one of the sulfur atoms occupies a 5'-bridging position. Representative dinucleotides possessing such a bond were synthesized by S-alkylation of nucleoside-3'-O-phosphorodithioates with 5'-halogeno-5'-deoxy-nucleosides. A fully protected dithymidylate containing internucleotide 5'-S-phosphorodithioate linkage was converted into a 3'-O-phosphoramidite derivative and employed for introduction of a modified dinucleotide into a predetermined position of the oligonucleotide sequence. The 5'-S-phosphorodithioate linkage in dinucleotide analogues was found to be resistant toward nucleolytic degradation with snake venom PDE and nuclease P1. However, P-stereoselective degradation was observed for diastereomers of 5'-S-phosphorodithioate dithymidine analogs under treatment with calf spleen PDE. The new 5'-S-phosphorodithioate linkage was readily degraded by iodine solutions in the presence of water. It was also found that oligothymidylates containing a single 5'-S-phosphorodithioate linkage form much weaker duplexes with their complementary sequences.

A method to evaluate a propidium iodide association with oligonucleotides and oligonucleotide-cationic lipid interactions.

Complex molecular ensembles are frequently considered an element of new pharmacological formulations. This is especially evident in the therapies based on genetic information. In order to obtain an effective drug, it is necessary to associate a nucleic acid molecule with the components to ensure the desired aggregate structure and properties. To evaluate the progress of the supromolecular aggregate formation a range of methodologies and techniques are needed to test the quality and uniformity of the formulations. In this paper we propose a procedure which measures the association of a small molecule with nucleic acid using propidium iodide and oligonucleotides as an example. To measure propidium iodide binding constant the oligonucleotide was covalently labeled with fluorescein and then the changes in fluorescence resonance energy transfer (FRET) were determined and handled according to the acceptor-donor titration methodology. The calculated binding constants were in a good agreement with the values published previously. The developed method was then used to evaluate the extent of an oligonucleotide association with the lipid aggregates. It was found that two populations of oligonucleotides are present in all lipid samples studied. The fraction of oligonucleotides associated with liposomes rises with the increase of a cationic lipid content, reaching the constant value when the fraction of cationic lipid exceeded 20 mol%. Energy transfer data combined with these obtained in quenching experiments show that the orientation of the oligonucleotide associated with a lipid bilayer depends on the amount of surface charge.

Inosylyl(3'-->5')inosine (IpI-). Acid-base and metal ion-binding properties of a dinucleoside monophosphate in aqueous solution.

The acidity constants of the (N7)H(+) sites of inosylyl(3'-->5')inosine (IpI(-)) were estimated and those of its (N1)H sites were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO3). The same method was used for the determination of the stability constants of the 1:1 complexes formed between Mg(2+), Co(2+), Ni(2+), Zn(2+), or Cd(2+) (= M(2+)) and (IpI - H)(2-) and, in the case of Mg(2+), also of (IpI - 2H)(3-). The stability constants of the M(IpI)(+) complexes were estimated. The acidity constants of H(inosine)(+) and the stability constants of the M(Ino)(2+) and M(Ino - H)(+) complexes were taken from the literature. The comparison of these and related data allows the conclusion that, in the M(IpI - H) species, chelates are formed; most likely they are preferably of an N7/N7 type. For the metal ions Co(2+), Ni(2+), Zn(2+), or Cd(2+), the formation degrees of the chelates are on the order of 60-80%; no chelates could be detected for the Mg(IpI - H) complexes. It is noteworthy that the (N1)H deprotonation, which leads to the M(IpI - H) species, occurs in all M(IpI)(+) complexes in the physiological pH range of about 7.5 or even below.

Discrimination in metal-ion binding to RNA dinucleotides with a non-bridging oxygen or sulfur in the phosphate diester link.

Replacement of a non-bridging oxygen in the phosphate diester bond by a sulfur has become quite popular in nucleic acid research and is often used as a probe, for example, in ribozymes, where the normally essential Mg(2+) is partly replaced by a thiophilic metal ion to reactivate the system. Despite these widely applied rescue experiments no detailed studies exist quantifying the affinity of metal ions to such terminal sulfur atoms. Therefore, we performed potentiometric pH titrations to determine the binding properties of pUp((S))U(3-) towards Mg(2+), Mn(2+), Zn(2+), Cd(2+), and Pb(2+), and compared these data with those previously obtained for the corresponding pUpU(3-) complexes. The primary binding site in both dinucleotides is the terminal phosphate group. Theoretically, also the formation of 10-membered chelates involving the terminal oxygen or sulfur atoms of the (thio)phosphate bridge is possible with both ligands. The results show that Mg(2+) and Mn(2+) exist as open (op) isomers binding to both dinucleotides only at the terminal phosphate group. Whereas Cd(pUpU)(-) only exists as Cd(pUpU)(-)(op), Cd(pUp((S))U)(-) is present to about 64 % as the S-coordinated macrochelate, Cd(pUp((S))U)(-)(cl/PS). Zn(2+) forms with pUp((S))U(3-) three isomeric species, that is, Zn(pUp((S))U)(-)(op), Zn(pUp((S))U)(-)(cl/PO), and Zn(pUp((S))U)(-)(cl/PS), which occur to about 33, 12 (O-bound), and 55 %, respectively. Pb(2+) forms the 10-membered chelate with both nucleotides involving only the terminal oxygen atoms of the (thio)phosphate bridge, that is, no indication of S binding was discovered in this case. Hence, Zn(2+) and Cd(2+) show pronounced thiophilic properties, whereas Mg(2+), Mn(2+), and Pb(2+) coordinate to the oxygen, macrochelate formation being of relevance with Pb(2+) only.

O-phenyl (triphenylphosphoniomethyl)phosphonate phenol solvate: supramolecular structure generated by O-H...O, C-H...O and C-H...pi(arene) hydrogen bonds.

The title compound, C(25)H(22)O(3)P(2).C(6)H(6)O, has a zwitterionic betaine-like structure and crystallizes as a phenol solvate. The two molecular components are held together by an almost linear intermolecular O-H...O hydrogen bond. The structure also contains three weak C-H...O and two C-H...pi(arene) interactions.

Metal-ion-coordinating properties of the dinucleotide 2'-deoxyguanylyl(5'-->3')-2'-deoxy-5'-guanylate (d(pGpG)3-): isomeric equilibria including macrochelated complexes relevant for nucleic acids.

The interaction between divalent metal ions and nucleic acids is well known, yet knowledge about the strength of binding of labile metal ions at the various sites is very scarce. We have therefore studied the stabilities of complexes formed between the nucleic acid model d(pGpG) and the essential metal ions Mg2+ and Zn2+ as well as with the generally toxic ions Cd2+ and Pb2+ by potentiometric pH titrations; all four ions are of relevance in ribozyme chemistry. A comparison of the present results with earlier data obtained for M(pUpU)- complexes allows the conclusion that phosphate-bound Mg2+ and Cd2+ form macrochelates by interaction with N7, whereas the also phosphate-coordinated Pb2+ forms a 10-membered chelate with the neighboring phosphate diester bridge. Zn2+ forms both types of chelates with formation degrees of about 91% and 2.4% for Zn[d(pGpG)]cl/N7 and Zn[d(pGpG)]-cl/PO, respectively; the open form with Zn2+ bound only to the terminal phosphate group, Zn[d(pGpG)]-op, amounts to about 6.8 %. The various intramolecular equilibria have also been quantified for the other metal ions. Zn2+, Cu2+, and Cd2+ also form macrochelates in the monoprotonated M[H;d(pGpG)] species (the proton being at the terminal phosphate group), that is, the metal ion at N7 interacts to some extent with the P(O)2(OH)- group. Thus, this study demonstrates that the coordinating properties of the various metal ions toward a pGpG unit in a nucleic acid differ: Mg2+, Zn2+, and Cd2+ have a significant tendency to bridge the distance between N7 and the phosphate group of a (d)GMP unit, although to various extents, whereas Pb2+ (and possibly Ca2+) prefer a pure phosphate coordination.

Acid-base properties of the nucleic-acid model 2'-deoxyguanylyl(5'-->3')-2'-deoxy-5'-guanylate, d(pGpG)3-, and of related guanine derivatives.

The dinucleotide d(pGpG) is an often employed DNA model to study various kinds of interactions between DNA and metal ions, but its acid-base properties were not yet described in detail. In this study the six deprotonation reactions of H4[d(pGpG)]+ are quantified. The acidity constants for the release of the first proton from the terminal P(O)(OH)2 group (pKa = 0.65) and for one of the (N7)H+ sites (pKa = 2.4) are estimated. The acidity constants of the remaining four deprotonation reactions were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO3): The pKa values for the deprotonations of the second (N7)H+, the P(O)2(OH)-, and the two (N1)H sites are 2.98, 6.56, 9.54 and 10.11, respectively. Based on these results we show how to estimate acidity constants for related systems that have not been studied, e.g. pGpG, which is involved in the initiation step of a rotavirus RNA polymerase. The relevance of our results for nucleic acids in general is briefly indicated.

Acid-base and metal-ion binding properties of the RNA dinucleotide uridylyl-(5'-->3')-[5']uridylate (pUpU3-).

It is well known that Mg2+ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote or inhibit catalytic activity. The ability of metal ions to coordinate simultaneously with two neighboring phosphate groups is important for ribozyme structure and activity. However, such an interaction has not yet been quantified. Here, we have performed potentiometric pH titrations to determine the acidity constants of the protonated dinucleotide H2(pUpU)-, as well as the binding properties of pUpU3- towards Mg2+, Mn2+, Cd2+, Zn2+, and Pb2+. Whereas Mg2+, Mn2+, and Cd2+ only bind to the more basic 5'-terminal phosphate group, Pb2+, and to a certain extent also Zn2+, show a remarkably enhanced stability of the [M(pUpU)]- complex. This can be attributed to the formation of a macrochelate by bridging the two phosphate groups within this dinucleotide by these metal ions. Such a macrochelate is also possible in an oligonucleotide, because the basic structural units are the same, despite the difference in charge. The formation degrees of the macrochelated species of [Zn(pUpU)]- and [Pb(pUpU)]- amount to around 25 and 90 %, respectively. These findings are important in the context of ribozyme and DNAzyme catalysis, and explain, for example, why the leadzyme could be selected in the first place, and why this artificial ribozyme is inhibited by other divalent metal ions, such as Mg2+.