[Spectral analysis and synthesis of artificial antigen of the organophosphors pesticide methamidophos].

In the present paper, a hapten of methamidophos was synthesized and conjugated with KLH by active ester method, thus the first artificial antigen was obtained. By diazotization method methamidophos conjugated with BSA, and the second artificial antigen was obtained. The synthesized haptens were characterized by MS, IR and 1H NMR, and the two artificial antigens were determined by the method of IR spectrum. The result implied that both the artificial antigens have absorbance peaks of hapten and protein, indicating that they were prepared successfully. This could provide evidence that the method of IR spectrum can be used to determine whether the artificial antigens are synthesized successfully.

Structure of a cardiotoxic phospholipase A(2) from Ophiophagus hannah with the "pancreatic loop".

The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

Structures of D-glyceraldehyde-3-phosphate dehydrogenase complexed with coenzyme analogues.

Crystal structures of GAPDH from Palinurus versicolor complexed with two coenzyme analogues, SNAD(+) and ADP-ribose, were determined by molecular replacement and refined at medium resolution to acceptable crystallographic factors and reasonable stereochemistry. ADP-ribose in the ADP-ribose-GAPDH complex adopts a rather extended conformation. The interactions between ADP-ribose and GAPDH are extensive and in a fashion dissimilar to the coenzyme NAD(+). This accounts for the strong inhibiting ability of ADP-ribose. The conformational changes induced by ADP-ribose binding are quite different to those induced by NAD(+) binding. This presumably explains the non-cooperative behaviour of the ADP-ribose binding. Unexpectedly, the SNAD(+)-GAPDH complex reveals pairwise asymmetry. The asymmetry is significant, including the SNAD(+) molecule, active-site structure and domain motion induced by the coenzyme analogue. In the yellow or red subunits [nomenclature of subunits is as in Buehner et al. (1974). J. Mol. Biol. 90, 25-49], SNAD(+) binds similarly, as does NAD(+) in holo-GAPDH. While, in the green or blue subunit, the SNAD(+) binds in a non-productive manner, resulting in a disordered thionicotinamide ring and rearranged active-site residues. The conformation seen in the yellow and red subunits of SNAD(+)-GAPDH is likely to represent the functional state of the enzyme complex in solution and thus accounts for the substrate activity of SNAD(+). A novel type of domain motion is observed for the binding of the coenzyme analogues to GAPDH. The possible conformational transitions involved in the coenzyme binding and the important role of the nicotinamide group are discussed.

Preliminary Crystallographic Analysis of Phospholipase A(2) from Naja naja kaouthia Lesson Venom.

An acidic phospholipase A(2) isolated from the venom of Naja naja kaouthia Lesson in Guangxi exhibits anticoagulative and hemolytic activities. In this work, the enzyme was crystallized by the method of hanging drop vapor diffusion. Two crystal forms were obtained and characterized by X-ray diffraction. One of them belonged to space group P 4 ( 3 ) 2 ( 1 )2 or P4(1)2(1)2 with unit cell parameters a b 8.797 nm, c 10.831 nm and there were three molecules per asymmetric unit the other belonged to space group P2(1)3 with unit cell parameters a b c 6.840 nm and there was one molecule per asymmetric unit. The diffraction data were collected up to 0.28 nm for each crystal form. The crystal properties of Naja naja verom phospholipase A(2) from different geographical regions are compared.

Structure Determination of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas in A New Monoclinic Crystal Form.

Basic phospholipase A(2) from the venom of Agkistrodon halys Pallas ( Agkistrodin blomhoffii brevicaudus ) exhibits hemolytic and anti-coagulant activities. A new monoclinic crystal form with four molecules per asymmetric unit was grown in the absence of n-octyl beta-o-glucopyranoside (beta-OG). The enzyme structure was determined by the molecular replacement method. The combined analysis of self- and cross- rotation function was used and non-crystallographic symmetry restraints were imposed to the structure refinement. The final model gave an acceptable crystallographic R factor and reasonable stereochemistry. Two molecules formed an interfacial-recognition-site linked dimer and two such dimers constituted a tetramer having pseudo 222 symmetry. Structural comparison with previously reported monoclinic forms, in which beta-OG was bound, showed that the variation of crystallization conditions had effects on the crystal packing, leading to significant changes of the cell parameters. Nevertheless, the structures of both the dimer and tetramer in the two crystal forms closely resembled to each other, indicating that the oligomers found in the monoclinic crystal forms were stable.