He-Chang Zou

Kinetics of Zn2+-induced Brain Type Creatine Kinase Unfolding and Aggregation

We studied the effect of Zn2+ on the folding and aggregation of brain creatine kinase (CK-BB). We developed a method to purify CK-BB from rabbit brain and conducted inhibition kinetics and unfolding studies of CK-BB. Zn2+ conspicuously aggregated and osmolytes, such as glycine and proline, were able to suppress the formation of aggregates and protect the enzymatic activity against Zn2+. These results suggest that Zn2+ might act as a risk factor for CK-BB in the brain under certain conditions, and some osmolytes may help CK-BB to sustain the active state when Zn2+ is present. Our study provides useful information regarding the effect of Zn2+ on brain-derived metabolic enzymes, especially those that are putatively related to brain disease. Furthermore, our study suggests that although Zn2+ may induce CK-BB inactivation and misfolding, the ability of some abundant proteins and osmolytes to chelate Zn2+ nonspecifically may protect CK-BB and allow it to exist in the active form.

The Effect of Zn2+ on Human Brain Creatine Kinase: Unfolding and Aggregation Studies

Abstract We studied the effects of Zn2+ on human brain creatine kinase (HBCK). Zn2+ inactivated the activity of HBCK in a dose dependent manner (IC50 = 0.06 mM). The time-interval kinetic studies showed that the inactivation followed first-order reaction kinetics with a biphasic process. The spectroflurorimetry results showed that Zn2+ conspicuously induced the tertiary structural change of HBCK with exposure of its hydrophobic surfaces. On the contrary, the secondary structure was slightly changed by Zn2+. We also found that HBCK aggregation was induced by Zn2+. This aggregation was dependent on the temperature and the enzyme and Zn2+ concentrations. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation and fully recover the conformation and activity of HBCK. Our study provides important insight into the unfavorable effect of Zn2+ on HBCK and it increases the understanding of the Zn2+ ligand-binding mechanism to the metabolic brain enzyme.

The effects of acrylamide on brain creatine kinase: Inhibition kinetics and computational docking simulation

The occurrence of acrylamide is frequently observed in processed foods. Therefore, the harmful effects of acrylamide on metabolic enzymes are important to understand. We studied the inhibitory effects of acrylamide on the brain creatine kinase (CK-BB). We found that CK-BB was kinetically inactivated by acrylamide accompanied by the disruption of the hydrophobic surface. Acrylamide mainly interacted with the thiol (-SH) residue of CK-BB and resulted in alkylation. A computational docking simulation supported that acrylamide directly bound to the active site of CK-BB where cysteine and glycine residues interacted mainly. The inhibition kinetics combined with computational prediction can be useful in order to have insights into the mechanisms regarding environmentally hazardous factors at the molecular level.

Effect of Cysteine Modification on Creatine Kinase Aggregation

We studied the effect of cysteine modification on creatine kinase (CK) aggregation as well as the kinetics of the process. We found that CK aggregation was modulated by different pH conditions in the presence of Zn2+, which is a CK aggregation trigger. The CK aggregation followed first-order kinetics, and this was effectively suppressed in acidic conditions. Even under the acidic condition, cysteine modification at the active site with using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) induced conspicuous aggregation in a dose-dependent manner. This aggregation process is directly related with decreasing the change of transition free-energy (ΔΔGAG). When dithiothreitol (DTT) was applied to the reaction system, the aggregates were significantly reduced: DTT treatment can fully reactivate (higher than 80%) the inactive CK that was separated from CK aggregates, whereas CK was completely inactivated by Zn2+ and DTNB. Some added osmolytes such as glycine and proline were able to successfully block CK aggregation by increasing the ΔΔGAG as well as by suppressing the hydrophobic CK surface. Our study suggests the effect of cysteine modification on the unfavorable aggregation of CK and on the aggregation process that followed first-order kinetics with the accompanying changes of transitional free energy and disruptions of the hydrophobic surface. We also demonstrate the successful protocol to block the aggregation.

Monomeric Creatine Kinase Aggregation and Sodium Dodecyl Sulfate-cyclodextrin Assisted Refolding

Abstract The monomelic state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a dénaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as α-, β-, and γ-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS- binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.

Effects of acrylamide on the activity and structure of human brain creatine kinase.

Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed.

Extraction and DNA digestion of 5′-phosphodiesterase from malt root

Abstract This study investigated the extraction of 5′-phosphodiesterase from malt root and the degradation of nucleic acids by this enzyme. The extraction used grade precipitation with ammonium sulfate and enzymatic hydrolysis. Samples were assayed using the modified Bradford method and high performance liquid chromatography. The results show that 5′-phosphodiesterase is isolated by grade precipitation with 30% and 80% saturation of ammonium sulfate and can be utilized to degrade deoxyribonucleic acid. The hydrolysate has four kinds of deoxynucleotides: 5′-dCMP, 5′-dTMP, 5′-dAMP, and 5′-dGMP. The optimum reaction temperature is 70°C, and the optimum pH is 5.5–6.0 for the reaction. The percentage of deoxynucleotides indicated by the China Pharmacopoeia (2000 edition) in the product is over 70%. The extraction of 5′-phosphodiesterase from malt root is shown to be possible and economical. Products from the enzymatic hydrolysate of DNA meet the pharmacopoeia.

The Effect of Ag+ on Arginine Kinase: Inhibition Kinetics

Abstract We studied the effects of Ag+ on arginine kinase (AK) from Fenneropenaeus chinensis. Ag+ inactivated the activity of AK in a dose dependent manner (IC 50 = 15 μM). Kinetic studies showed that the inactivation of AK by Ag+ was reversible and occurred in a noncompetitive inhibition manner (K i = 2.8 μM). Spectroflurorimetry results showed that Ag+ did not induce conspicuous tertiary structural changes in AK at the corresponding concentration ranges of inactivation studies. However, the secondary structure measured by circular dichroism was slightly changed by Ag+. Taken together, these data suggest that the active site of AK is flexible, with the complete loss of activity occurring prior to significant changes in overall structures. Our study provides important insight into the inhibitory mechanism of Ag+ on AK and increases our understanding of the influence of Ag+ on the mechanism of this metabolic enzyme.

Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate

Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions.