Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.     

Isolation of melatonin by immunoaffinity chromatography

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.     

Purification of thiogalactoside transacetylase by affinity chromatography

Thiogalactoside transacetylase, the product of the lacA gene of the lactose operon of Escherichia coli, has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration.     

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Purification of alternanase by affinity chromatography

The enzyme alternanase, produced by Bacillus sp. NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating α(1→6), α(1→3) linkages. The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties. An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme. Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure. When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering. The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield. Electronic Publication     

High pressure liquid chromatography of conjugated gibberellins

The application of high pressure liquid chromatography to the purification and identification of conjugated gibberellins was examined. Two kinds of reversed phase columns, octadesylsilanized and dimethylsilanized silica gel, were useful for the isolation and identification of gibberellin A₃ glucoside and gibberellenic acid glucoside from immature seeds of Quamoclit pennata.     

Immunoaffinity chromatography

The basic procedure of immunoaffinity chromatography (IAC) is described. The insoluble support matrices available for IAC and their activation chemistries, including some of the most recently introduced, are reviewed. Means of selecting the most appropriate monoclonal antibody (MAb) are described, although an empirical approach is still required for the final choice of antibody. Precise methods of runing IAC columns are surveyed including the binding, washing, and elution stages, although no precise recommendations can be made particularly for the elution step since this is unique to a particular MAb and antigen. All IAC sorbents lose activity with time through a combination of MAb inactivation and ligand leakage. The relative importance of the two phenomena is discussed, and suggestions are made to minimize the problem along with an indication of the relative stabilities of a range of coupling chemistries. A sample of the proteins purified by IAC is given together with pointers to the future of the technique.     

Chiral counter-current chromatography: A survey of its instrument,mechanism, procedure,and applications

The chiral separation by counter-current chromatography has made great progress in the past three decades. It has become increasingly popular in the field of chiral separation, and many applications have been introduced during the last years. This review mainly focuses on the current topics, applications, and trends in chiral separation by counter-current chromatography. It contains the development of modern counter-current chromatography apparatus, theory of counter-current chromatography, overview of applications of chiral counter-current chromatography enantioseparation, its current situation, and challenges. At last, some conclusions and perspectives also have been discussed in this review.     

Countercurrent gradient chromatography: A continuous focusing technique

The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (>2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. (c) 1995 John Wiley & Sons, Inc.     

Separation of enkephalin degradation products by ligand-exchange chromatography

Enkephalin degradation products can be accurately analyzed by cascade chromatography through XAD polystyrene and copper-Chelex columns. One of the main degradation products generated during the incubation of enkephalin with rat striatal membranes, the N-terminal amino acid Tyr, is adsorbed quantitatively on the copper-Chelex columns whereas the minor, but probably specific, product TyrGlyGly is not.     

Thin-layer chromatography of thiosulfonate anions

Organic thiosulfonates of the form RS(O₂)S^? can be separated from the characteristic contaminants in synthetic preparations by thin-layer chromatography on silica gel. Thiosulfonates and sulfinates can both be visualized, and differentiated, by treatment of plates with a FeCl₃ reagent in acetone. Data are presented for seven aromatic and six aliphatic thiosulfonate anions and the corresponding sulfonates and sulfinates. With exception of the series related to cysteic acid, R_f values are always in the order RS(O)₂S^? > RSO₃^? > RSO₂^?.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

Purification of muscle uridine diphosphoglucose pyrophosphorylase by hydrophobic chromatography

Uridine diphosphoglucose pyrophosphorylase was purified 2500-fold from rabbit skeletal muscle with a total recovery of 35% of the initial activity. The present procedure was made possible by an extensive use of hydrophobic chromatography. Purified pyrophosphorylase had a specific activity of 500 mumol/min/mg of protein and was homogeneous by chromatographic and electrophoretic criteria. The enzyme appears to be composed of eight subunits of 53,000 molecular weight each.     

Affinity chromatography of estrogen receptors on diethylstilbestrol-agarose

Diethylstilbestrol was coupled to epoxy-activated agarose yielding an affinity resin which is highly efficient for the isolation of estrogen receptors. This resin, diethylstilbestrol-agarose (DES-agarose), bound two proteins (Mr = 50,000 and 65,000) from rabbit uterine cytosol that show a specific interaction with estradiol. A two step procedure--adsorption on DES-agarose followed by a selective elution with p-sec-amylphenol and NaSCN, yielded highly purified estrogen receptors which can be used in the studies of estradiol-receptor interactions with other cell constituents.     

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.     

Adsorption chromatography of proteins

A method for adsorption chromatography of proteins is proposed. A protein solution is passed through a cellulose column at a pH value corresponding to an isoelectric point of the protein. Depending on the charge of unwanted proteins, they either remain at the origin (if charges of protein and ion-exchanger are opposite) or are released from the column (if charges of protein and ion-exchanger coincide). Elution volume of the purified protein is higher than for the second group of unwanted proteins because movement of the uncharged protein of interest includes its adsorption on cellulose followed by subsequent desorption caused by the elution buffer. Problems of optimization of buffers and adsorbents are discussed. Applicability of the method of adsorption chromatography is illustrated using purification of horseradish peroxidase as an example.     

Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.