Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra :CH 2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 K d protein and a noninteracting 1.7 K d peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13 CH 2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

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l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3 IU/mg protein.

The advantages of using superdisintegrants, sodium starch glycolate (SSG), croscarmellose sodium (CCS) and crospovidone (CP) over traditional high molecular weight (MW) viscosity enhancing agents, guar gum (GG), xanthan gum (XG), pectin and high MW HPMC are examined for improving drug content uniformity without compromising dissolution of films containing nanoparticles of griseofulvin (GF), used as a model poorly water-soluble drug. Films were fabricated by preparing low MW HPMC solutions to which a fixed amount of viscosity enhancing agents were added and mixed with GF nanosuspensions produced via wet milling, followed by casting and drying. The addition of superdisintegrants and high MW HPMC, led to an increase in viscosity of precursor suspensions without GF particle aggregation, and hence excellent drug content uniformity along with retention of the high surface area of the GF nanoparticles in dried films. In contrast, addition of XG and pectin resulted in aggregation of GF particles in suspensions, leading to poor content uniformity and incomplete recovery of GF nanoparticles upon redispersion of dried films. In spite of their high precursor viscosity, the films containing superdisintegrants did not lead to increased mechanical strength and demonstrated fast drug release, suggesting faster matrix erosion. In contrast, films with high MW polymers (GG, XG, and pectin and high MW HPMC) had increased mechanical strength and their subsequent slow erosion/disintegration along with longer hydration times resulted in significant delay of drug release, which was found to be directly proportional to their MW. These results demonstrate novel use for superdisintegrants as economical and superior alternative to traditional viscosity enhancing agents in forming drug laden biocompatible polymer films.