Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.     

The biosynthetic gene cluster for a monocyclic beta-lactam antibiotic, nocardicin A

The monocyclic beta-lactam antibiotic nocardicin A is related structurally and biologically to the bicyclic beta-lactams comprised of penicillins/cephalosporins, clavams, and carbapenems. Biosynthetic gene clusters are known for each of the latter, but not for monocyclic beta-lactams. A previously cloned gene encoding an enzyme specific to the biosynthetic pathway was used to isolate the nocardicin A cluster from Nocardia uniformis. Sequence analysis revealed the presence of 14 open reading frames involved in antibiotic production, resistance, and export. Among these are a two-protein nonribosomal peptide synthetase system, p-hydroxyphenylglycine biosynthetic genes, an S-adenosylmethionine-dependent 3-amino-3-carboxypropyl transferase (Nat), and a cytochrome P450. Gene disruption mutants of Nat, as well as an activation domain of the NRPS system, led to loss of nocardicin A formation. Several enzymes involved in antibiotic biosynthesis were heterologously overproduced, and biochemical characterization confirmed their proposed activities.     

Chitinase and beta-N-acetylglycosaminidase in the digestive juice of Helix pomatia.

A beta-N-acetylglucosaminidase from Helix pomatia digestive juice was separated and partly purified by gel chromatography. The optimal pH for the degradation of p-nitrophenyl-N-acetyl-beta-D-glucosaminide was 3.4. The molecular weight was around 160 000 and the pI = 4.95. In the same gel chromatography run two chitinase active peaks were also obtained. These chitinase active peaks were also obtained. These chitinases, with molecular weights around 26 000 and 13 000, had somewhat different pH activity curves with optima at 4.2 and 4.3. By isoelectric focusing the first peak with molecular weight around 26 000 was divided in two chitinase active regions with pI at 5.7 and 3.5. The second peak with molecular weight around 13 000 had a pI at 7.3.     

Electrochemically controlled hydrogen bonding. Nitrobenzenes as simple redox-dependent receptors for arylureas

Reduction of nitrobenzene derivatives in the presence of arylureas in aprotic solvents results in large positive shifts in potential of the nitrobenzene(0/)(-) cyclic voltammetry wave with little change in wave shape. This behavior is indicative of reversible hydrogen bonding between nitrobenzene radical anions and arylureas. Computer fitting of the cyclic voltammetry of 4-nitroaniline, NA, plus 1,3-diphenylurea in DMF shows essentially no binding between urea and NA in the oxidized state (K(ox) < 1 M(-)(1)), but very strong binding in the reduced state (K(red) = 8 x 10(4) M(-)(1)), along with very rapid rates of hydrogen bond formation (k(f)'s approximately 10(8)-10(10) M(-)(1) s(-)(1)), making this system a fast on/off redox switch.     

Abstract,weighted, and multidimensional Adamjan-Arov-Krein theorems,and the singular numbers of Sarason commutants

The classical Adamjan-Arov-Krein (A-A-K) theorem relating the singular numbers of Hankel operators to best approximations of their symbols by rational functions is given an abstract version. This provides results for Hankel operators acting in weightedH ²(T; ), as well as inH ²(T ^d ), and an A-A-K type extension of Sarason's interpolation theorem. In particular, it is shown that all compact Hankel operators inH ²(T ^d ) are zero.Author partially supported by NSF grant DMS89-11717.     

Comparison of protein adsorption patterns onto differently charged hydrophilic superparamagnetic iron oxide particles obtained in vitro and ex vivo.

Protein adsorption patterns of superparamagnetic iron oxides (SPIO) were evaluated by two-dimensional electrophoresis (2-DE) after in vitro incubation of the particles in plasma or serum. SPIO particles having positive (MKK 1211), negative (MKA 1211), or neutral (MKG 1411) charge were used. Protein adsorption patterns of different charged SPIO particles acquired in vitro and recollected 5 min after intravenous injection into rats (ex vivo) were compared. For the uncharged MKG 1411 particles, the differences of protein adsorption patterns were negligible and only minor differences were found for the negatively charged MKA 1211 and positively charged MKK 1211 particles. A good correlation between in vitro and ex vivo data could be shown. For the evaluation of protein adsorption patterns of SPIO particles determining organ distribution and allowing estimation of site-specific delivery (drug targeting), the currently used protocol for 2-DE analysis could be confirmed.     

Isothermal Photo Differential Scanning Calorimetry. Crosslinking polymerization of multifunctional monomers in presence of visible light photoinitiators

The effect of photoinitating (camphorquinone/amines) system concentration and sample thickness on the photopolymerization of triethyleneglycol dimethacrylate/2-hydroxypropyl methacrylate has been investigated. The rate of photopolymerization reaches a maximum and then decreases as the concentration of photoinitiating system increases. This effect has been intepreted as inner filter effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

On the moment of inertia in deformed Ba-Xe nuclei as deduced from gamma-gamma energy correlation experiments

The γ-rays following reactions induced by bombarding targets of ^{114, 116, 118, 120, 122}Sn with 118 MeV ¹²C ions were investigated using six NaI(Tl) detectors in a two-dimensional coincidence arrangement. Experimental energy-correlation spectra were extracted from the original coincidence matrices. The energy-correlation spectra exhibit the features expected for rotational nuclei and were used to deduce information on the moment of inertia $\text{I}{}^{\mathrm{(2)}}\text{= ΔI/Δω}$. The gross properties of the behaviour of $\text{I}$⁽²⁾ in the Ba-Xe region are discussed together with their interpretation within the cranked shell model (CSM).     

Some applications of the use of the reflectance photodensitometer to spot test analysis

Summary The colour developed during certain spot tests has been measured using a reflectance photodensitometer. In this way a quantitative estimation of the amount of substances present in the test solution has been made. The relationship between densitometer reading and concentration of test substance, and the best method of obtaining uniform colour in the spots are discussed. Several substances have been analysed by this method, for which only 5 to 10l of solution are required. 5 to 0.2 g of metallic ions, 100 to 5g of polyamide and 1 to 0.05 micro-equivalents of amino nitrogen can be determined. Precision compares well with other colorimetric methods.

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Zusammenfassung Die bei einigen Tüpfelreaktionen entwickelte Farbe wurde mit einem Reflexions-Photodensitometer gemessen. Dies ermöglicht eine quantitative Beurteilung der in der Probelösung vorhandenen Menge Substanz. Das VerhÄltnis zwischen den Densitometerablesungen und der Konzentration der Probelösung sowie die beste Methode der Herstellung gleichmÄßig gefÄrbter Tüpfelflecke werden besprochen. Mehrere Substanzen wurden mit Hilfe dieser nur 5 bis 10l benötigenden Methode untersucht. 5 bis 0,2g eines Metall-Ions, 100 bis 5g eines Polyamids und 1 bis 0,05 Mikro-Val Amino-Stickstoff können bestimmt werden. Ein Vergleich der Genauigkeit der Methode mit der anderer kolorimetrischer Methoden fÄllt vorteilhaft aus.

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Résumé A l'aide d'un photodensimètre à réflexion, on a mesuré la couleur qui apparaÎt au cours de certains essais à la touche. Il a été ainsi possible d'estimer quantitativement les teneurs de substances présentes dans des solutions soumises à l'étude. Les relations existant entre les lectures densitométriques et les concentrations des substances étudiées sont discutées par l'auteur ainsi que la meilleure méthode d'obtention de taches de couleurs uniformes. Plusieurs substances ont été analysées à l'aide de cette méthode qui ne nécessite que 5 à 10l de solution; on peut ainsi doser 5 à 0,2g d'ions métalliques, 100 à 5g de polyamides et 1 à 0,05 microéquivalents d'azote aminé. La précision obtenue est comparable à celle des autres méthodes colorimétriques.