A promiscuous glutathione transferase transformed into a selective thiolester hydrolase

Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.     

Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography

A new downstream procedure for the isolation of bovine lactoferrin (bLf), lactoperoxidase and bovine lactoferricin (LfcinB) from sweet cheese whey was developed at the laboratory scale, based on membrane adsorber technology. The procedure was upscaled later on to an industrially relevant scale for the purificationof sweet whey concentrate with a recovery yield for lactoferrin of more than 90%. Based on these results the industrial process for 1 x 10(8) kg whey per year was projected. These high-value proteins were downstreamed by using cation-exchange membrane systems (Sartobind S, Sartorius, G?ttingen, Germany). These strongly acidic membranes trap proteins in its anionic form. The dynamic loading capacity for both proteins as well as the optimal elution profiles with sodium chloride gradients were derived from laboratory experiments using membrane modules with 15-75 cm2 membrane material. Further investigations were performed with 1 m2 modules in a continuous process mode. The enzymatic preparation of LfcinB from bLf was performed by pepsin hydrolysis and the isolation of LfcinB was directly carried out from the enzymatic digest mixture. The identification of the proteins was performed with matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS). LfcinB and bLf were both tested afterwards in biological assays in order to show not only the efficiency of the downstreaming process in regard to product quantity but also to product quality (biological activity).     

Microarray technology as a universal tool for high-throughput analysis of biological systems

Over the last years microarray technology has become one of the principal platform technologies for the high-throughput analysis of biological systems. Starting with the construction of first DNA microarrays in the 1990s, microarray technology has flourished in the last years and many different new formats have been developed. Peptide and protein microarrays are now applied for the elucidation of interaction partners, modification sites and enzyme substrates. Antibody microarrays are envisaged to be of high importance for the high-throughput determination of protein abundances in translational profiling approaches. First cell microarrays have been constructed to transform microarray technology from an in vitro technology to an in vivo functional analysis tool. All of these approaches share a common prerequisite: the solid support on which they are generated. The demands on this solid support are thereby as manifold as the applications themselves. This review is aimed to display the recent developments in surface chemistry and derivatization, and to summarize the latest developments in the different application areas of microarray technology.     

Cubature over the sphere in Sobolev spaces of arbitrary order

This paper studies numerical integration (or cubature) over the unit sphere for functions in arbitrary Sobolev spaces H^s(S²), s>1. We discuss sequences of cubature rules, where (i) the rule Q_m(n) uses m(n) points and is assumed to integrate exactly all (spherical) polynomials of degree ≤n and (ii) the sequence (Q_m(n)) satisfies a certain local regularity property. This local regularity property is automatically satisfied if each Q_m(n) has positive weights. It is shown that for functions in the unit ball of the Sobolev space H^s(S²), s>1, the worst-case cubature error has the order of convergence O(n^-s), a result previously known only for the particular case . The crucial step in the extension to general s>1 is a novel representation of , where P_ℓ is the Legendre polynomial of degree ℓ, in which the dominant term is a polynomial of degree n, which is therefore integrated exactly by the rule Q_m(n). The order of convergence O(n^-s) is optimal for sequences (Q_m(n)) of cubature rules with properties (i) and (ii) if Q_m(n) uses m(n)=O(n²) points.     

A lower bound for the worst-case cubature error on spheres of arbitrary dimension

This paper is concerned with numerical integration on the unit sphere S^r of dimension r≥2 in the Euclidean space ℝ^r+1. We consider the worst-case cubature error, denoted by E(Q_m;H^s(S^r)), of an arbitrary m-point cubature rule Q_m for functions in the unit ball of the Sobolev space H^s(S^r), where s>, and show that The positive constant c_s,r in the estimate depends only on the sphere dimension r≥2 and the index s of the Sobolev space H^s(S^r). This result was previously only known for r=2, in which case the estimate is order optimal. The method of proof is constructive: we construct for each Q_m a `bad' function f_m, that is, a function which vanishes in all nodes of the cubature rule and for which Our proof uses a packing of the sphere S^r with spherical caps, as well as an interpolation result between Sobolev spaces of different indices.     

The <Emphasis Type="Italic">s</Emphasis>-energy of spherical designs on <Emphasis Type="Italic">S</Emphasis><Superscript>2</Superscript>

This paper investigates the s-energy of (finite and infinite) well separated sequences of spherical designs on the unit sphere S ². A spherical n-design is a point set on S ² that gives rise to an equal weight cubature rule which is exact for all spherical polynomials of degree ≤n. The s-energy E _s (X) of a point set of m distinct points is the sum of the potential for all pairs of distinct points . A sequence Ξ = {X _m } of point sets X _m ⊂S ², where X _m has the cardinality card(X _m )=m, is well separated if for each pair of distinct points , where the constant λ is independent of m and X _m . For all s>0, we derive upper bounds in terms of orders of n and m(n) of the s-energy E _s (X _m(n)) for well separated sequences Ξ = {X _m(n)} of spherical n-designs X _m(n) with card(X _m(n))=m(n).      

Fatty acid binding sites of human and bovine albumins: Differences observed by spin probe ESR

Bovine and human serum albumins and recombinant human albumin, all non-covalently complexed with 5- and 16-doxyl stearic acids, were investigated by ESR spectroscopy in solution over a range of pH values (5.5–8.0) and temperatures (25–50 °C), with respect to the allocation and mobility of fatty acid (FA) molecules bound to the proteins and conformation of the binding sites. In all proteins bound FA undergo a permanent intra-albumin migration between the binding sites and inter-domain residence. Nature identity of the recombinant human albumin to its serum-derived analog was observed. However, the binding sites of bovine albumin appeared shorter in length and wider in diameter than those of human albumin. Presumably, less tightly folded domains in bovine albumin allow better penetration of water molecules in the interior of the globule that resulted in higher activation energy of FA dissociation from the binding site. Thus, the sensitive technique based on ESR non-covalent spin labeling allowed quantitative analysis and reliable comparison of the fine features of binding proteins.     

Zeros of linear combinations of Laguerre polynomials from different sequences

We study interlacing properties of the zeros of two types of linear combinations of Laguerre polynomials with different parameters, namely and . Proofs and numerical counterexamples are given in situations where the zeros of R_n, and S_n, respectively, interlace (or do not in general) with the zeros of , , k=n or n−1. The results we prove hold for continuous, as well as integral, shifts of the parameter α.     

Convexity of the zeros of some orthogonal polynomials and related functions

We study convexity properties of the zeros of some special functions that follow from the convexity theorem of Sturm. We prove results on the intervals of convexity for the zeros of Laguerre, Jacobi and ultraspherical polynomials, as well as functions related to them, using transformations under which the zeros remain unchanged. We give upper as well as lower bounds for the distance between consecutive zeros in several cases.