Weak Anion Exchange Chromatography Research Articles

Purification and identification of the heat-stable factor required for pregnenolone-binding protein activity. Evidence that the factor is adenosine 3‘,5‘-diphosphate.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% α-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated α (fast) and β (slow) based on electrophoretic mobility under denaturing conditions. The ratio of α to β chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosin. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the α and β chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an αβ hetero-dimer.

Multiple forms of peptidyl alpha-amidating enzyme: purification from rat medullary thyroid carcinoma CA-77 cell-conditioned medium.

Conditioned medium from cultures of rat medullary thyroid carcinoma CA-77 cells was used as a source for purification of the secreted forms of peptidyl alpha-amidating enzyme. The alpha-amidating enzyme activity was partially purified using a combination of weak anion exchange and gel filtration chromatography. Subsequent strong anion exchange chromatography at pH 6.0 resolved this partially pure enzyme into four distinct peaks of activity, termed Ia, Ib, II, and III. Peaks Ia and Ib exhibited broad pH optima between pH 6.0-8.5, whereas peaks II and III both exhibited pH optima at approximately pH 5.0. The peak III activity was further purified to electrophoretic homogeneity using hydrophobic interactive chromatography followed by strong anion exchange chromatography at pH 8.0. The enzyme exhibited an apparent molecular mass of 75K, a pH optimum of approximately pH 5.0, and a maximal turnover number of 580 min-1 in the presence of L-ascorbate. Kinetic studies demonstrated that the enzyme probably functions through a ping-pong mechanism with respect to the binding of the glycine-extended peptide substrate and the L-ascorbate cofactor. The peak III enzyme exhibits several distinctive characteristics compared to amidating enzymes isolated and characterized by other laboratories.

Linear multidimensional liquid chromatography in the preparative scale purification of calmodulin from brain extract

Rapid preparative scale purification of calmodulin from crude bovine brain extract is achieved in a single chromatographic run by physically coupling two different liquid chromatography columns which employ different separation mechanisms. In this case columns packed with newly commercialized 40-μm silica-based hydrophobic interaction and 5-μm micron silica-based weak anion-exchange chromatography media were used. The only sample preparation required for conducting this purification procedure is the addition of salt to the crude brain supernatant to promote the initial binding of calmodulin to the hydrophobic interaction chromatography media. Chromatography carried out on such linear arrangements of columns has been referred to as linear multidimensional liquid chromatography.

1] High-performance liquid chromatographic separation of ascorbic acid, erythorbic acid, dehydroascorbic acid, dehydroerythorbic acid, diketogulonic acid, and diketogluconic acid

Publisher Summary This chapter emphasizes the separation of L-ascorbic acid (AA) and D-erythorbic acid (EA) from their respective oxidation and hydrolysis products by weak anion-exchange high- performance liquid chromatography (HPLC). The primary basis for the physiological roles of vitamin C is the oxidation–reduction system between AA and L-dehydroascorbic acid (DHAA). EA is epimeric to AA at C-5 and possesses little or no vitamin C activity; it may be an antagonist of vitamin C. The high-performance liquid chromatography (HPLC) separation of AA, EA, L-diketogulonic acid (DKGulA), and D-diketogluconic (DKGluA) on aminopropyl bonded-phase silica is accomplished by weak anion-exchange chromatography with acetonitrile–0.05 M KH 2 PO 4 eluant. The conjugated enediol chromophore in AA and EA allows their detection at 270 nm; reduction of DHAA and D-dehydroerythorbic acid (DHEA) with dithiothreitol (DTT) allows their indirect determination from the increase in AA and EA absorption. In aqueous solution, DHAA exists as a hydrated bicyclic hemiketal and in DKGulA both ketone functions are hydrated. Refractive index detection is the method of choice for the compounds considered, especially with the recent development of highly sensitive detectors.

Separation and purification of diastereomers of angiotensin I by weak anion-exchange high-performance liquid chromatography

Several diasteroemers of angiotensin I were resolved by weak anion-exchange high-performance liquid chromatography (HPLC). All of the diastereomers which were examined contained significant amounts of peptides whose amino acid composition differed from the designated diastereomer of angiotensin I. The des-Asp 1 forms of angiotensin I and the impurities were weakly retained on the anion-exchange column and were well separated from the rest of the peptides. Many of the impurities contained fractional amounts of amino acids suggesting that peptides which contained variable amounts of histidine and arginine are not resolved by this method. The results are compared with the results of separations of the same peptides by reversed-phase HPLC. This comparison strongly suggests that the two HPLC methods, utilizing different separation principles, are complementary; hence their combined use leads to a more confident assessment of the purity of a given peptide preparation.

Weak anion-exchange high-performance liquid chromatography of peptides

In this survey, the principles and applications of a method recently developed for peptide separations are given. This method uses a bonded weak anion-exchange column and mixtures of volatile triethylammonium acetate buffer and acetonitrile as eluent. Its applications to the separation of a large number of peptides including diastereomeric and other closely related peptides are discussed. Separation of the enzymatic digests of some proteins is also presented. The complementary use of this method to the reversed-phase methods is outlined and their combined use for separation of enzymatic digests of proteins and assessment of purity of synthesized peptides is demonstrated. The results reviewed show that the weak anion-exchange method is an excellent approach for peptide separations and could be an important partner of reversed-phase methods for achieving optimal results.