Quantitative determination of deferiprone in human plasma by reverse phase high performance liquid chromatography and its application to pharmacokinetic study.

Deferiprone (1, 2 dimethyl-3-hydroxypyrid-4-one) is considered to be the standard iron chelator. Pharmacokinetic studies of generic formulations are required in local condition before placed on the market. High performance liquid chromatographic (HPLC) method was used for quantification of deferiprone in human plasma using UV/VIS detector. Chromatographic separation was carried out on C(18) column, with a mobile phase of methanol-buffer (18:82, v/v), pH 3.5, and caffeine was used as an internal standard. The calibration curve was linear over the range 0.25-10 µg/mL in human plasma (R(2) = 0.9994). After oral administration of deferiprone (500 mg) to human, the plasma concentration-time curve of deferiprone was conformed to two-compartment open model. The deferiprone plasma concentration showed a rapid absorption and average area under the plasma concentration-time curve (AUC) of deferiprone was 17.0 ± 1.23 h.µg/ml. Average absorption and elimination half-life values of deferiprone of 24 volunteers were 0.62 ± 0.12 and 2.65 ± 0.43 hours. This study confirms the rapid absorption of deferiprone in humans. AUC was similar to that previously reported but C(max) was slightly lower than that stated in the literature.

Chemical modification results in hyperactivation and thermostabilization of Fusarium solani glucoamylase.

Chemical modification of carboxyl groups of glucoamylase from a mesophilic fungus, Fusarium solani, was carried out using ethylenediamine as nucleophile in the presence of water-soluble 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Modification brought about a dramatic enhancement of catalytic activity and thermal stability of glucoamylase. Temperature and pH optima of ethylenediamine-coupled glucoamylase (ECG) increased as compared with those of native enzyme. The specificity constant (k(cat)/K(m)) of native, ECG-2, ECG-11, and ECG-17 was 136, 173, 225, and 170, respectively, at 55 degrees C. The enthalpy of activation (Delta H*) and free energy of activation (Delta G*) for soluble starch hydrolysis were lower for the chemically modified forms. All of the modified forms were stable at higher temperatures and possessed high Delta G* against thermal unfolding. The effects of alpha-chymotrypsin and subtilisin on the modified forms were activating as compared with native. Moreover, denaturation of ECG-2, ECG-11, and ECG-17 in urea at 4 mol x L(-1) also showed an activation trend. A possible explanation for the thermal denaturation of native and increased thermal stability of ECG-2, ECG-11, and ECG-17 at higher temperatures is also discussed.

Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase.

Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and pK (a)'s of active site residues. The specificity constants (K (cat)/K (m)) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55 degrees C for starch, respectively. The enthalpy of activation (DeltaH*) and free energy of activation (DeltaG*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by alpha-chymotrypsin and subtilisin resulted in activation.

Studies on kinetics and thermostability of a novel acid invertase from Fusarium solani.

The present investigation deals with purification and thermal characterization of an acid invertase produced by Fusarium solani in submerged culture. The maximum enzyme activity (9.90 U mL(-1)) was achieved after 96 h of cultivation at pH 5.0 and 30 degrees C in a basal medium containing molasses (2%) as the carbon and energy source supplemented with 1% peptone. Invertase was purified by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephadex G-200. The purified enzyme was proven to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the enzyme was 65 kDa. The optimum pH and temperature for activity were 2.6 and 50 degrees C, respectively. The Km value for sucrose was 3.57 mM with an activation energy of 4.056 kJ mol(-1). Enthalpies of activation (DeltaH) were decreased while entropies (DeltaS) of activation increased at higher temperatures. The effects of alpha-chymotrypsin and 4 M urea were tetraphasic with periodic gain and loss of enzyme activity. A possible explanation for the thermal inactivation of invertase at higher temperatures is also discussed.