A deadenylase assay by size-exclusion chromatography

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.     

Refolding of denatured thioredoxin observed by size-exclusion chromatography

Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance size-exclusion chromatography of peptides

Gel filtration on soft gels has been employed for over 40 years for the separation, desalting and molecular weight estimation of peptides and proteins. Technical improvements have given rise to high-performance size-exclusion chromatography (HPSEC) on rigid supports, giving more rapid run times and increased resolution. Initially, these packings were more suitable for the separation of proteins than of peptides, but supports that operate in the fractionation range <10,000 Daltons (Da) are now available. In this report, HPSEC is described in relation to its application to peptides, especially regarding purification, estimation of molecular weight and study of molecular associations.     

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Background

The number of species with completed genomes, including those with evidence for recent whole genome duplication events has exploded. The recently sequenced Atlantic salmon genome has been through two rounds of whole genome duplication since the divergence of teleost fish from the lineage that led to amniotes. This quadrupoling of the number of potential genes has led to complex patterns of retention and loss among gene families.

Results

Methods have been developed to characterize the interplay of duplicate gene retention processes across both whole genome duplication events and additional smaller scale duplication events. Further, gene expression divergence data has become available as well for Atlantic salmon and the closely related, pre-whole genome duplication pike and methods to describe expression divergence are also presented. These methods for the characterization of duplicate gene retention and gene expression divergence that have been applied to salmon are described.

Conclusions

With the growth in available genomic and functional data, the opportunities to extract functional inference from large scale duplicates using comparative methods have expanded dramatically. Recently developed methods that further this inference for duplicated genes have been described.

     

Measurement of osmotic second virial coefficients by zonal size-exclusion chromatography

Numerical simulation of protein migration reflecting linear concentration dependence of the partition isotherm has been used to invalidate a published procedure for measuring osmotic second virial coefficients (B₂₂) by zonal exclusion chromatography. Failure of the zonal procedure to emulate its frontal chromatographic counterpart reflects ambiguity about the solute concentration that should be used to replace the applied concentration in the rigorous quantitative expression for frontal migration; the recommended use of the peak concentration in the eluted zone is incorrect on theoretical grounds. Furthermore, the claim for its validation on empirical grounds has been traced to the use of inappropriate B₂₂ magnitudes as the standards against which the experimentally derived values were being tested.     

Rapid characterization of cellular retinoid-binding proteins by high-performance size-exclusion chromatography

A high-performance size-exclusion chromatographic method was developed for the analysis of cellular retinol and retinoic acid-binding proteins. The chromatographic analysis of the retinol-retinol-binding protein complex or the retinoic acid-retinoic acid-binding protein complex requires 15 min. The use of high-specific-radioactivity retinoid ligand (30-40 Ci/mmol) allows routine detection of 25 fmol of retinoid-binding protein/mg of cytosolic protein. Thus, this method provides a rapid alternative to sucrose density gradient sedimentation analysis of the cellular retinoid-binding proteins. High-performance size-exclusion chromatography is well suited to screening novel tissues, tumors, and cell lines for the presence of retinoid-binding proteins and to the further characterization of these cellular proteins. This method was applied to the characterization of cellular retinol- and retinoic acid-binding proteins in fetal rabbit tissues. Both retinol- and retinoic acid-binding proteins were detected in fetal rabbit brain, intestine, kidney, and lung at a gestational age of 28 days. Neither retinoid-binding protein was detected in 28-day-old fetal rabbit placenta. Specific retinoic acid binding in fetal intestinal cytosol decreased as a function of increasing gestational age.     

Preparative and analytical size-exclusion chromatography of chitosans

Two chitosan samples (fraction of acetylated units (F_A) 0.15 and 0.52) were fractionated by preparative size exclusion chromatography (SEC). The molecular weights and molecular weight distributions of the fractions were analyzed by analytical size exclusion chromatography coupled to an on-line low angle laser light scattering detector and a differential refractive index detector (SEC-LALLS-DRI), and their intrinsic viscosities were determined. The exponent (a) of the Mark-Houwink-Kuhn-Sakurada (MHKS) equation was found to be 0.92 ± 0.07 and 1.1 ± 0.1, respectively, at I = 0.1 and pH 4.5. No variation in F_A related to molecular weight was found. Reversible interaction between chitosans and different column packings strongly influenced the log M-V relationships. This interaction was generally most pronounced for the low-F_A chitosan, suggesting that the protonated amino groups are involved. Ammonium acetate buffer reduced this effect and the use of a new type of SEC-packing seemed to eliminate it. The more highly acetylated chitosan also had a more pronounced tendency towards concentration dependent self-association, which most probably involve intermolecular hydrophobic interactions between the acetyl groups.     

High-performance size-exclusion chromatography of hydrolyzed plant proteins

A high-performance size-exclusion chromatographic (SEC) procedure has been developed to determine the molecular size distribution of several sources of hydrolyzed plant proteins including soy, potato, cotton seed, corn gluten, wheat bran, and wheat germ. The SEC packing consisted of a glycerylpropylsilyl layer covalently bonded to 100-Å pore-size silica particles (10 μm). A number of mobile phases were evaluated in an attempt to reduce adsorption between hydrolyzed components and packing material. Systems containing either sodium dodecyl sulfate or methanol showed the best performances; however, adsorption could not totally be eliminated. The combination of size-exclusion and retention mechanisms of separation was valuable in establishing differences among samples. Based on dialysis of hydrolyzed soy protein, it appeared that most of the adsorbed material was of low molecular weight. Column recovery studies indicated complete elution of samples.     

Immunoassay-based determination of phenobarbital using size-exclusion chromatography

The measurement of the anti-epileptic drug phenobarbital from serum samples combining immunoassay and size-exclusion chromatography is presented. The immunoreaction is based on the competitive binding of the analyte (unlabelled phenobarbital) and the fluorescent-labelled phenobarbital to anti-phenobarbital antibodies. Mixing of the reagents and the immunoreaction takes place in a flow system. The products are separated on-line on a short gel chromatographic column and the fluorescence intensity of the marker is measured. The calibration curve shows good linearity in the range 5–80 μg/ml, corresponding to therapeutically relevant serum levels. Intra-day precision values are between 7.32 and 9.48%; the accuracy is between 0.97 and 9.43%. Inter-day precision and accuracy measured on 6 different days fall between 5.38 and 10.05% and −8.27 and −4.97%, respectively. The results obtained with the proposed method show a good correlation with those of other methods (radioimmunoassay and fluorescence polarisation immunoassay) already established in clinical laboratories.     

分子筛层析法分析重组乙肝表面抗原聚集物的形成

分子筛层析作为分析蛋白质颗粒聚集物的一种有力工具,被用于研究重组乙肝表面抗原聚集物的形成。已去除聚集物的表面抗原放置在不同的理化条件下或经过不同的纯化方法处理后,应用HPLC分析其聚集物的形成。为研究发酵过程中是否形成表面抗原聚集物,酵母细胞破碎后立即用Sepharose 4 FF层析柱分离为不同的组分,并分别进行HPLC分析。结果发现,在纯化过程和酵母发酵阶段都有表面抗原聚集物的产生。     

Characterization of polysaccharide-protein complexes by size-exclusion chromatography combined with three detectors

Two water-soluble samples (TM1 and TM2) were extracted from Pleurotus tuber-regium using 0.9% aqueous NaCl at 20 and 80 degrees C to obtain relatively low molecular mass fractions. The chemical structure of TM1 was analyzed to be a branched heteropolysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan containing galactose and mannose. TM2 was a branched polysaccharide-protein complex, and the sugar moiety was mainly beta-(1-->6)-, beta-(1-->4)-, and beta-(1-->3)-linked glucan. Preparative size-exclusion chromatography (SEC) and analytical SEC combined with three detectors were used to detect the TM1 and TM2 samples, confirming that the proteins were bonded to the polysaccharides. Furthermore, analytical SEC combined with online laser light scattering, differential refractive index detector, and UV to determine the components, weight-average molecular mass (M(w)) and chain conformation of the samples. The relatively low exponent values (nu) of S(2)(z)(1/2)=k(nu)M(w)(nu) for the samples in 0.15M aqueous NaCl at 25 degrees C suggested that TM1 and TM2 existed in compact sphere conformation in the aqueous solution. This work provided valuable information on the structure and chain conformation characterization of the polysaccharide-protein complex having relatively low M(w).     

Some potentialities and drawbacks of contemporary size-exclusion chromatography

The present state of the chromatographic techniques based on differentiation of solutes according to their molecular sizes is briefly surveyed. Attention is centred on high-performance techniques applied to purification and characterization of natural macromolecules, and on discussion of the chromatographic approaches to the determination of the molecular masses and molecular mass distributions of both natural and synthetic polymers. The basic requirements on the selection of the separation system and the experimental conditions are summarized, demonstrated on a few examples and critically evaluated.     

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.     

Protein refolding at high concentration using size-exclusion chromatography

A new method to improve refolding yields and to increase the concentration of refolded proteins in a single operation has been developed. The method uses size-exclusion chromatography matrices to perform buffer exchange, aggregate removal, and the folding reaction. The reduced diffusion of proteins in gel-filtration media has been shown to suppress the nonspecific interactions of partially folded molecules, thus reducing aggregation. Hen egg white lysozyme (HEWL) and bovine carbonic anhydrase (CAB) were successfully refolded from initial protein concentrations of up to 80 mg/mL using Sephacryl S-100 (HR). The aggregation reaction for lysozyme was reduced and was only detected at the highest protein concentration used. The average recovery of lysozyme was 63%, with an average specific activity of 104%. Carbonic anhydrase experiments also showed that aggregation was suppressed and the average protein recovery from the column was 56%, with a specific activity of 81%. This process enables refolding and the purification of active species to be achieved in a single step. (c) 1996 John Wiley & Sons, Inc.     

Purification of cell culture-derived human influenza A virus by size-exclusion and anion-exchange chromatography

A process comprising of size-exclusion chromatography (SEC) and anion-exchange chromatography (AEC) was investigated for downstream processing of cell culture-derived influenza A virus. Human influenza virus A/PR/8/34 (H1N1) was propagated in serum-free medium using MDCK cells as a host. Concentrates of the virus were prepared from clarified and inactivated cell culture supernatants by cross-flow ultrafiltration as described before. SEC on Sepharose 4 FF resulted in average product yields of 85% based on hemagglutination (HA) activity. Productivity was maximized to 0.15 column volumes (cv) of concentrate per hour yielding a reduction in total protein and host cell DNA (hcDNA) to 35 and 34%, respectively. AEC on Sepharose Q XL was used to separate hcDNA from virus at a salt concentration of 0.65 M sodium chloride. Product yields >80% were achieved for loads >160 kHAU/mL of resin. The reduction in hcDNA was 67-fold. Split peak elution and bimodal particle volume distributions suggested aggregation of virions. Co-elution with hcDNA and constant amounts of hcDNA per dose indiciated association of virions to hcDNA. An overall product yield of 52% was achieved. Total protein was reduced more than 19-fold; hcDNA more than 500-fold by the process. Estimation of the dose volume from HA activity predicted a protein content at the limit for human vaccines. Reduction of hcDNA was found insufficient (about 500 ng per dose) requiring further optimization of AEC or additional purification steps. All operations were selected to be scalable and independent of the virus strain rendering the process suitable for vaccine production.     

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.     

Separation of proanthocyanidins by degree of polymerization by means of size-exclusion chromatography and related techniques

The molecular masses of polyphenols in plants and food vary greatly up to the order of 10 kDa. Polymerized polyphenols are not only natural antioxidants but also strong inhibitors of numerous physiological enzymatic activities. Several useful methods for the determination and separation of these high-molecular-mass polyphenols have recently been developed. In this review, details of the methods and applications of size-exclusion chromatographic separation of polymerized polyphenols, particularly those of proanthocyanidins, are described and compared with other related chromatographic or mass spectrometric analyses.     

Macromolecular distribution of Acacia senegal gum (gum arabic) by size-exclusion chromatography

Fractionation of Acacia senegal gum has been carried out on Sephacryl gels S-400 and S-500. The shape and relative height of the chromatograms are sample dependent.Physicochemical data including circular dichroism and the weight-average molecular weights distribution show that about 70% of the material is composed of homogeneous polysaccharide chains with a very low nitrogen content. The remaining material is a combination of polysaccharide and nitrogen moieties (probably an arabinogalactan-protein complex).Our data are consistent with the new and simplified structural models proposed recently for this polysaccharide.