Microcalorimetric Studies On Biological Macromolecules And Mimetic Biomineralization Of Calcium Oxalate

Based on the basic principles and methods of microcalorimetry, by using LKB-2107 BATCH microcalorimeter system, thermokinetic studies on Na~+,K~+-ATPase enzymatic reaction are carried out; and then, researches on the thermodynamic aspects of rare earth ions (III) binding to bovine serum albumin are made; finally, companion microcalorimetry to X-ray diffraction and scanning electromicroscopy, the mimetic biomineralization of is discussed. The detailed studies are listed as bell v:I Microcalorimetric studies on Na~+,K~+-ATPase and enzyme inhibition by ATP1. For the first time, the ATP hydrolysis reaction catalyzed by Na~+,K~+-ATPase under mimetic physiological conditions (310.15K, pH7.4, ATP in millimolar concentration range) was studied with microcalorimetry. The Michaelis-Menton Constant (K_m) and Maximum Velocity (V_max) of Na~+, K~+-ATPase under given experimental conditions were determined by reduced extent method. The obtained values of K_m and F_max were (0.479±0.020) ×10~-3 mol L~-1, 0.681 ± 0.026 umol P_imin~-1 mg protein~-1, respectively. The reliability of the data was further confirmed by colorimetric studies.2. Given a series of initial substrate concentrations, the apparent molar reaction enthalpy △_rH_m of Na~+,K~+-ATPase at 310.15K and pH7.4 was measured as -40.514±0.9 k J mol~-1.3. The effect of Na~+,K~+-ATPase pre-dilution on its activities was investigated. Results showed that excessive pre-dilution greatly decreases the enzyme activities. It is suggested that there occurred dilution inactivation phenomenon on Na~+,K~+-ATPase. It was concluded that the active heterodimer form were probably destroyed by excessive pre-dilution.4. Under mimetic physiological conditions (310.15K, pH7.4, ATP in millimolar concentration range), the inhibition kinetics of Na~+,K~+-ATPase by ATP was deeply studied. Results showed that MgATP complex was the real substrate with a K_m value of about 0.5mM and free ATP was a competitive inhibitor with a K\ value of 0.253mM.II Studies on the interactions of Bovine Serum Albumin (BSA) with rare earth ions or drug molecule1. Under mimetic physiological conditions (310.15K, pH7.4, 0.1mol/L NaCl), the thermodynamic aspects of a series of rare earth ions (III) binding to bovine serum albuminwere studied on LKB-2107 BATCH microcalorimeter. By the calorimetric results, the numbers (n\, n2) and the molar enthalpies (Ar#mi, Ar//m2) of different binding sites (high-affinity and low-affinity, respectively) were determined directly for the first time. Results showed that the enthalpies are all positive ones, that is to say the binding of rare earth ions (III) to BSA are all entropy-driven reactions. Furthermore, by using spectrofluorometry, the number (?i) and the apparent binding constants (lgA^i) of high-affinity binding sites were obtained. By UV-different spectrum, the binding position of rare earth ions (III) on BSA were characterized.2. Enoxacin[l-ethyl-6-fluoro-l,4-dihydro-4-oxo-7-(l-piperazinyl)-l,8-naphthyridine-3-carboxyl-ic acid] is a representative one of the third kinds of fluoroquinolone antibiotics. By spectrofluorometry, the interaction between enoxacin and bovine serum albumin (BSA) in aqueous solution under given conditions (7M5°C/25°C/37°C, pH=7.0, O.lmol/L NaCl) was studied. Using fluorescence quenching method, the binding distance (r) between enoxacin and BSA was determined. The obtained r values were 2.019 nm at 15°C, 2.054 nm at 25°C, 2.040 nm at 37°C, respectively. The dissociation constants (Kn) of drug-BSA were determined as 1.09 X 10"5, 1.48 X10"5, 1.97 X 10"5M, respectively. According to the thermodynamic parameters, it was inferred that electric action was the main force. It is useful for the clinic. And the effects of competition between the drug and coexisting rare earth ions for BSA were investigated for the first time. Results showed that the competition effects of rare earth ions weaken the interaction between drug and protein. It was found that the dependence of the dissociation constants (Ku) on the atomic number of rare earth ions (Zre) exhibits "tetrad effect" and "gadolinium break", across the series.IQ Studies on the mimetic biomineralization of calcium oxalate (CaCjO^ and the control effects of rare earth ions1. By using microcalorimetry, the kinetics of CaCaCU crystallization in pure water system and different PC-H2O systems were studied. Moreover, the crystal structure, morphology, and the shape of CaC2C>4 obtained in different systems were investigated by X-ray diffraction, scanning electromicroscopy and polarized microscopy for revealing the effects of the reaction systems' ordered state on the nucleation, growth, aggregation, crystal structure, and the morphologies of CaC2O4. It can be useful for opening out the principles of the urinary stones' biomineralization. Further, the effects of different reaction conditions on the crystallization of CaC2O4 were investigated. Results showed that the kinetic parameters of CaC2O4 crystal growth varied for different reaction systems, thecrystal structure and morphology dependent on the aggregation state of PC in systems. In conclusion, the results obtained revealed a complex dependence of calcium oxalate nucleation, crystal growth, aggregation, structure and morphology on the state of reaction systems and the detailed conditions. The observed phenomena such as the inhibition effect of crystal aggregation in PC-H2O systems, the control of crystal structure and morphology by PC ordered systems, are helpful for the cure of urinary stones from the biomedical point of view.2. By microcalorimetry, the effects of rare earth ions (RE3+) on the kinetics of CaC2C<4 crystallization in aqueous solutions were investigated. Moreover, the control effects of RE3+ on crystal structure, morphology, and the size of CaC2C>4 were studied by X-ray diffraction and SEM. Further, the effects of La3+ on the crystallization kinetics, crystal structure, morphology, and size of CaCiCM in PC-H2O lipsome ordered-system were studied. The conclusions obtained were listed as follows:(1) The microcalorimetric results showed that the effects of RE3+ not only on the initial nucleation of CaC2C?4 but also on the crystal growth, and the optimal inhibition effects on nucleation and crystal growth can only obtain at appropriate concentrations.(2) The X-ray diffraction results showed that under given conditions, the obtained CaC2O4 crystal form from pure water system is merely monohydrate (COM), however, the additions of RE3+ favor the produce of dihydrate (COD) and COD will be the exclusive crystal form at certain concentrations of RE3+.(3) The SEM results showed that the additions of RE3+ cause significant controls on the crystal shape, size and dissolution properties of CaC2O4, and crystals with different morphologies, different sizes will be obtained as the concentrations of RE3+ varies.(4) The results about the effects of La3+ in aqueous system and PC-H2O liposome ordered-system showed that there are differences in the detailed effects of La3+ in the two different systems. It is assumed that this may be caused by the difference of micro reaction-environment.(5) In conclusion, the inhibition effects of RE3+ on the crystal nucleation, crystal growth, and the controls on the crystal structures, morphologies, sizes, dissolution properties of CaC2O4, such as favoring the produce of COD, smaller-size spherical crystal, promote the dissolution of crystal, all are helpful for the prevention and therapy of urinary stones.