Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-ActivatedTM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid–base hydrolysis. Kinetic parameters (Km and Vmax) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

The effect of the microstructure on the phase behavior of mixtures of polybutadiene (PB) and poly(ethylene-co-1-butene) (PEB) with polystyrene (PS) has been investigated. A series of PBs with 1,2-addition content ranging from 7 to 93% were synthesized by anionic polymerization, and a portion of each was subsequently hydrogenated to yield PEB. Polymer pairs with blend compositions from 10 to 90 wt % were cast from toluene for each of the 16 PS/PB pairs and 7 PS/PEB pairs. Laser light scattering was used to obtain cloud point measurements, which were then used to construct phase diagrams. It was found that, for constituent components with equivalent degrees of polymerization, PS/PEB pairs give rise to higher upper critical solution temperatures than PS/PB pairs, indicating that PS/PEB pairs are less miscible than PS/PB pairs. Experimental phase diagrams were curve-fitted to theoretical phase diagrams predicted from the Flory−Huggins theory with the expression for the interaction parameter α:  α = a + b/T + cφPS/T, where α is related to the Flory−Huggins interaction parameter χ by χ = αV r, where V r is the molar reference volume, T is the absolute temperature, and φPS is the volume fraction of PS in the mixture. α values for PS/PB mixtures increase with increasing 1,2-addition (miscibility decreases) while α values for PS/PEB mixtures decrease (miscibility increases) with increasing 1-butene content. Using these α values, the Helfand−Wasserman theory was applied to predict the order−disorder transition temperatures of PS-block-PB and PS-block-PEB copolymers with varying 1,2-addition and 1-butene content, respectively.

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS3 (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the posttranslational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138 - Cys154, Cys143 - Cys150, Cys282 - Cys396, Cys470 - Cys482, Cys471 - Cys484, and Cys475 - Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (> 70%). A successful methodology has been developed which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

Examination, by optical microscopy, of the matrix/analyte morphology of a sample prepared from a droplet in matrix-assisted UV laser-desorption mass spectrometry (MUVLDMS) reveals that the matrix crystallizes to a large extent, thus altering the concentration of the material in regions on the surface from that of the bulk applied solution. Microscopy reveals that vanillic acid and to a lesser extent nicotinic acid matrices form prominent crystalline rings around the dried sample spot while sinapic acid forms a more uniform dispersion of the crystallized matrix. Insulin analyte investigated in this report tends to form a thin isotropic phase in the center of the drops. The first reported use of a KrF excimer laser (249 nm) in MUVLDMS is also discussed.