Two different crystal forms of pig pancrleatic a-amylase isoenzyme I1 (PPAII), free and complexed to a carbohydrate inhibitor (acarbose), have been compared together and to previously reported structures of PPAI. A crystal form obtained at 4"C, containing nearly 72% solvent, made it possible to obtain a new complex with acarbose, different from a previous one obtained at 20°C [Qian, M., Buisson, G., DuCe, E., Haser, H. & Payan, F. (1994) Biocrbemistry 33, 6284-62941. In the present form, six contiguous subsites of the enzyme active site are occupied by the carbohydrate ligand; the structural data indicate that the binding site is capable of holding more than the five glucose units of the scheme proposed through kinetic studies. A monosaccharide ring bridging two protein molecules related by the crystal packing is located on the surface, at a distaince of 2.0 nm from the reducing end of the inhibitor ligand; the symmetry-related glucose ring in the crystal lattice is found 1.5 nm away from the non-reducing end of the inhibitor ligand.

Pig pancreatic a-amylase (PPA, E.C. 3.2. I. 17, 496 amino-acid residues) has been crystallized as a complex with a lectin-like inhibitor from bean Phaseolus vulgaris (224 amino-acid residues tbr the inhibitor monomer). The hanging-drop vapour-diffusion method was used to grow crystals from solutions containing 2-methyl-2,4-pentanediol as precipitant. The crystals belong to monoclinic space group C2 with a :o152.5, b= 80.3, c= 68.8A,, /~= 91.4 and diffract to 2.9A resolution. A molecular-replacement solution of the structure has been obtained using the refined PPA and LoLl (Lathyrus ochrus isolectin I) atomic coordinates as starting models. Low-resolution refinement of the model is underway. The analysis reveals that the functional inhibitor molecule is dimeric and interacts with two molecules of enzyme.

Since food contact materials (FCMs) are in direct contact in consumer products, the existence of extractables and leachables in the packaging and storage materials can raise health and safety concerns. To address these concerns and comply to the regulations, companies demand methods and techniques to analyze their products for research and development as well as quality assurance purposes. In response to this increasing demand, we developed a method to analyze extractables and leachables in FCMs using a liquid chromatography high accuracy mass spectrometer.Common food storage products including plastic bag, plastic cup, and aluminum foil were selected to be analyzed. Equal size strips from each sample were extracted in water, 3% acetic acid, 10% ethanol, and 95% ethanol. Blank was collected in every matching conditions for comparisons. A Restek Viva C18 column (100 x 2.1 mm, 3 µm) was used for chromatography. Mobile phase A was water with formic acid and ammonium acetate additives. Mobile phase B was acetonitrile. A generic gradient from 30% to 100% B was used and the total run time was 15 min. A Shimadzu LCMS-9030 Quadrupole time-of-flight mass spectrometer was used in scan mode (100-2000 m/z) for the possible extractable and leachable identification.Potential extractable and leachable compounds were isolated by comparing the extracted samples and extracted blanks. Formula predictor was used to identify compounds of interest according to accurate mass and isotopic pattern. Unknown ion 282.2784 m/z at retention time 7.6 min was identified as oleamide, a common slip agent in polyethylene films. Unknown ion 340.3564 m/z at retention time 10.2 min was identified as docosanamide, a releasing agent for PVC, polyolefin, polystyrene and other plastic. Unknown ion 338.3413 m/z at retention time 9.1 min was identified as cis-13-docosenoamide, another releasing agent for plastic. MS/MS spectra for each of the potential extractable and leachable compounds were obtained and matched with open source databases such as Metlin. Highly similar fragment patterns between experimental unknowns and database were observed which further supports the identification results.

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