α-Crystallin Assisted Refolding of Enzyme Substrates: Optimization of External Parameters

α-Crystallin is known to act as a molecular chaperone by preventing the aggregation of partially unfolded substrate proteins. It is also known to assist the refolding of a number of denatured enzymes, but the activity yield is often less than 20%. In this paper, we have tried to tune the refolding ability of α-crystallin in vitro by optimizing various external parameters. We wanted to find out the best possible condition under which it can exhibit maximum refolding capacity. We found that under suitable condition in vitro α-crystallin can refold denatured malate dehydrogenase, carbonic anhydrase and lactate dehydrogenase to recover more than 40% activity. We also measured the effect of several external factors such as nucleotides, osmolytes, electrolytes, temperature etc. on the in vitro α-crystallin mediated reactivation of above stated enzymes. We found that nucleotides and electrolytes had little effect on the refolding ability of α-crystallin. However, in presence of different osmolytes, we found that its ability to reactivate denatured substrate proteins enhanced significantly. Refolding in presence of pre-incubated α-crystallin reveals that hydrophobicity had stronger influence on the refolding capacity of α-crystallin than its oligomeric size.     

Carbonic Anhydrase Ⅱ : Structures,Functions and Inhibitors

Human carbonic anhydrase Ⅱ is one of the most efficient one in carbonic anhydrase isozymes, which catalyzes the reversible hydration dehydration of CO2 and water : CO₂ + H₂O\ HCO₃^- + H⁺. It is found in virtually every tissueand cell type, and involves in many human physiological and pathological processes, such as human acid-base balance, glaucoma, osteoporosis, and cancer. Since discovered in 1940, carbonic anhydrase Ⅱ has been an important drug target with more attention. Up to now, many inhibitors were discovered including some clinical therapeutic drugs. This paper reviewed recent developments in structures, functions and inhibitors of human carbonic anhydrase Ⅱ.     

Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase Ⅱ in Hepatocellular Carcinoma

Early detection and intervention are key strategies to reduce mortality, increase longterm survival, and improve the therapeutic effects of hepatocellular carcinoma(HCC) patients.Herein, the isobaric tag for relative and absolute quantitation(iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues,surrounding noncancerous tissues, and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC. In total, 22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues, respectively, compared with the distal noncancerous tissues. Among these proteins, carbonic anhydrase II(CA2) was identified to be significantly upregulated in the secretome of cancerous tissues; correspondingly, the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations. Interestingly, a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence, and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival. Regarding the mechanism, the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway. Taken together, this study identified a novel biomarker for HCC diagnosis and prognosis, and provided a valuable resource of HCC secretome for investigating serological biomarkers.     

Factors Controlling Induction of External Carbonic Anhydrase and Change in K?(CO2) of Photosynthesis in Chlorella regularis

Transfer of algal cells of Chlorella regularis from 3% CO₂ inair into ordinary air in the light increased external carbonicanhydrase (CA) activity as well as photosynthetic affinity forCO₂ by several-fold within 2 h. Since no noticeable differencewas observed in CA activity between intact cells and cell homogenates,CA seemed to be mainly localized on the cell surface. Changesin CA activity and K_?(CO₂) of photosynthesis were not observedin the dark. CA induction was 50%-inhibited by incubation with10 µM DCMU during adaptation of high-CO₂ cells to air,whereas it was considerably suppressed when high-CO₂ cells preincubatedwith DCMU in the light for 6 h or without DCMU in the dark for24 h were used. The change in K_?(CO₂) of photosynthesis wasonly slightly affected by DCMU. Uncoupler like carbonylcyanide-m-chlorophenyl-hydrazone(CCCP) and inhibitors of mitochondrial respiration (KCN plussalicylhydroxamic acid) suppressed CA induction during adaptationof high-CO₂ cells to low CO₂ conditions. These results suggest that photosynthesis is not essential forCA induction in Chlorella regularis when some amounts of photosyntheticproducts are previously stored in the cells and respirationis active. A decrease in K_?(CO₂) of photosynthesis during adaptationfrom high to low CO₂ was mostly independent on photosynthesis.However, light is essential for both phenomena. (Received July 16, 1990; Accepted January 21, 1991)     

Solid-phase peptide synthesis and circular dichroism study of chiral β-peptoid homooligomers

Summary. N-alkyl-β-alanine oligomers (β-peptoids) with α-chiral side chains [(R)- or (S)-1-(phenylethyl)amino groups] were synthesized and analyzed by CD spectroscopy. These chiral β-peptoid homomers exhibited chain-length-dependent and solvent-dependent ellipticity, strongly indicating the presence of a secondary structure in solution. The CD behaviour was only slightly temperature-dependent upon heating, as also previously observed for stable α-peptoid helices containing the same type of side chains. Thus, the data presented here comprise the first evidence for a chain length-dependent secondary folding of compounds with this novel peptidomimetic backbone design. In addition, applicability of a novel hyphenated technique, HPLC-SPE-NMR/MS, for analysis of crude SPPS reaction products was demonstrated. Authors’ address: Dr. C. A. Olsen, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark; Present address: The Scripps Research Institute     

Impacts of Elevated CO2 Concentration on Biochemical Composition, Carbonic Anhydrase, and Nitrate Reductase Activity of Freshwater Green Algae

To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186μmol/L and the biochemical composition, carbonic anhydrase (CA),and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate,and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186μmol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in ChloreUa pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186μmol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The &#103 -glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond &#103 -glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond &#103 -glucosidase. The substrate specificity of the almond &#103 -glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond &#103 -glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

N-aryl-N’-ureido-O-sulfamates: Potent and selective inhibitors of the human Carbonic Anhydrase VII isoform with neuropathic pain relieving properties

Herein we report for the first time an efficient synthetic procedure for the preparation of N-aryl-N’-ureido-O-sulfamates (AUSs) as a new class of Carbonic Anhydrase Inhibitors (CAIs). The compounds were tested for the inhibition of several human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) isoforms. Interesting inhibition activity and high selectivity against CA VII and XII versus CA I and II, with K_Is in the low nanomolar range, were observed. Molecular modeling studies allowed us to decipher the structural features underpinning the selective inhibitory profile of AUSs towards isoforms CAs VII and XII. A selection of sulfamates showed promising neuropathic pain modulating effects in an in vivo animal model of oxaliplatin induced pain.     

A Widespread Silent Polymorphism of Human Carbonic Anhydrase III (31 Ile ? Val): Implications for Evolutionary Genetics

During amino acid sequence studies of carbonic anhydrase (CA) III, purified from a pool of human skeletal muscles, an electrophoretically undetectable (silent) variation was found at residue 31 which was either valine and/or isoleucine. To distinguish a simple allelic polymorphism from more complex models involving gene duplication, 11 separate CA III samples were purified from individuals of different age and racial backgrounds. Peptide mapping by high performance liquid chromatography and sequencing indicated that four were homozygous for 31-Val, three homozygous for 31-Ile and four were apparent heterozygotes. Since the ratio of Val/Ile at residue 31 was approximately 1.0 in the heterozygotes, the present observations are consistent with a simple allelic polymorphism model. Despite the small sample size, there are preliminary indications that the gene frequencies may differ among racial groups. The finding of this silent allelic polymorphism together with the finding of an electrophoretically detectable polymorphism of CA II permits us to test the linkage of the CA II and CA III genes which appear to have been formed by gene dupliction more than 300 million years ago. The possibility that the Val/Ile variation may represent a neutral mutation is discussed.     

Artificial Chaperone-assisted Refolding of GuHCl-Denatured α-Amylase at Low Temperature: Refolding versus Aggregation

Refolding of GuHCl-denatured α-amylase was investigated using the artificial chaperone-assisted method. Three different cationic detergents (CTAB, TTAB and DTAB) and two nonionic detergents (Tween 80 and Triton X-100) were evaluated as the capturing reagents along with α- and β-CD as the stripping agents. The refolding yields, at a final protein concentration of 0.15 mg/ml, were 82, 71 and 66% in the presence of β-CD and CTAB, TTAB or DTAB, respectively. To improve the refolding yield and to suppress the extent of aggregation, the initial rate of the stripping step was slowed down by maintaining the refolding environment at 4°C for about 3 min followed by raising the temperature to 25°C. Under this thermal procedure, the refolding yield and the extent of aggregation were changed from 82 and 25% at 25°C to 94 and 7% at 4°C, respectively. These findings may assist the activity recovery of recombinant proteins at relatively high concentrations.     

Identification of β-Lactamase Inhibitory Peptide Using Yeast Two-Hybrid System

Random oligonucleotide fragments were designed and amplified by PCR and fused with the activating domain of pGAD424 to construct a random peptide library. The DNA fragment encoding beta-lactamase was fused with the binding domain of pGBT9(+2). Subsequently, using yeast two-hybrid system we found two positive clones encoding peptides P1 and P2 that have the ability to bind beta-lactamase in vivo. The genes encoding P1 and P2 were cloned into pGEX-4T-1. GST-peptide fusion proteins were expressed in Escherichia coli and isolated by glutathione-Sepharose 4B affinity chromatography. Finally, P1 and P2 were cleaved from the fusion protein with thrombin and purified by ultrafiltration. Inhibition assay of peptides with beta-lactamase in vitro indicated that only P1 has the ability to inhibit beta-lactamase.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

The Important Role of Active Site Water in the Catalytic Mechanism of Human Carbonic Anhydrase II – A Semiempirical MO Approach to the Hydration of CO2 †

The approach of CO₂ to a series of active site model complexes of human carbonic anhydrase II (HCAII) and its catalytic hydration to bicarbonate anion have been investigated using semiempirical MO theory (AM1). The results show that direct nucleophilic attack of zinc-bound hydroxide to the substrate carbon occurs in each model system. Further rearrangement of the bicarbonate complex thus formed via a rotation-like movement of the bicarbonate ligand can only be found in active site model systems that include at least one additional water molecule. Further refinement of the model complex by adding a methanol molecule to mimic Thr-199 makes this process almost activationless. The formation of the final bicarbonate complex by an internal (intramolecular) proton transfer is only possible in the simplest of all model systems, namely {[Im₃Zn(OH)]⁺·CO₂}. The energy of activation for this process, however, is 36.8 kcal·mol^–1 and thus too high for enzymatic catalysis. Therefore, we conclude that within the limitations of the model systems presented and the level of theory employed, the overall mechanism for the formation of the bicarbonate complex comprises an initial direct nucleophilic attack of zinc-bound hydroxide to carbon dioxide followed by a rotation-like rearrangement of the bicarbonate ligand via a penta-coordinate Zn²⁺ transition state structure, including the participation of an extra active site water molecule.Electronic Supplementary Material available.     

Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. -Cyclodextrin–acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.Revisions received 29 September 2004     

Prediction of the β-Hairpins in Proteins Using Support Vector Machine

By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting β-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 β-hairpins. The overall accuracy of prediction and Matthew’s correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew’s correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154–159). The performance of the method is also evaluated by predicting the β-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.     

Nano-coating of β-galactosidase onto the Surface of Lactose by Using an Ultrasound-assisted Technique

We nano-coated powdered lactose particles with the enzyme β-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached β-galactosidase was measured through its enzymatic reaction product D-galactose using a standardized method. A near-linear increase was obtained in the thickness of the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for β-galactosidase, did not undergo enzymatic degradation during processing and remained unchanged for at least 1 month. Stability of protein-coated lactose was due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder using a simple approach.     

Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.