Zafar H. Zaidi

Purification and characterization of a chymotrypsin Kunitz inhibitor type of polypeptide fro the venom of cobra (Naja naja naja)

A chymotrypsin Kunitz inhibitor type of polypeptide has been isolated from the venom of Naja naja naja by reverse phase HPLC and cation exchange FPLC. It is present in a considerably lower amount than that of the corresponding trypsin inhibitor. The primary structure, determined by sequence analysis of the whole molecule and its tryptic peptides, has 57 residues with an apparent molecular mass of 6.2 kDa. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. Of residues considered functionally important in Kunitz‐type inhibitors, Gly‐36 is replaced by Ser in a segment of weak contacts with the protease.

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N‐terminal region. A fragment representing an N‐terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom.

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.

Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A2 (PLA2-H1)

A toxic phospholipase A2 (PLA2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle fibers and direct histopathological examination of the injected muscles (i.m.). PLA2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular infiltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The purified PLA2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty infiltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked infiltration of inflammatory cells within alveoli. Our results suggest that the purified PLA2-H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria.

Extensive multiplicity of the miscellaneous type of neurotoxins from the venom of the cobra Naja naja naja and structural characterization of major components

A multiplicity of miscellaneous type neurotoxins were detected in the venom of the cobra Naja naja naja by use of reverse‐phase HPLC and FPLC. The primary structures of major forms were determined, giving 4 novel structures. All four contain 62–65 residues, with 10 half‐cystine residues and resemble the miscellaneous type of toxins from other Naja species. Differences within the species are extensive, exchanges occur at 27 positions, giving only 58% residue identity between all forms. However, the differences are largely limited to 3 regions corresponding to structurally important loops where two functional residues participating in receptor binding are exchanged. The four miscellaneous neurotoxins now characterized, together with the minor components of the miscellaneous type, the minimally four neurotoxins reported before, and other related toxins, indicate the existence of an extensive toxin gene multiplicity.

Pharmacological effects of the leaf-nosed viper snake (Eristocophis macmahoni) venom and its HPLC fractions

Crude venom from Eristocophis macmahoni was demonstrated to exert a potent inhibition of human blood platelet aggregation mediated by adenosine diphosphate (ADP), platelet activating factor (PAF) and arachidonic acid (AA). The venom caused lysis of the platelets, however, the red blood cells were not lysed by the venom. Substantial oedema was produced upon injection of the venom into the rat hind paw. Contrarily, the intraperitoneal injection of the venom to the rats caused an inhibition of the carrageenin-induced rat paw oedema. However, an 100% lethality within 24 h was observed with a dose of 40 mg/kg body weight. The venom was fractionated by reverse phase high pressure liquid chromatography (HPLC) and the fractions were analyzed for their effect on ADP-induced platelet aggregation. The fraction eluted at 15.5 min (20% acetonitrile concentration) exhibited an inhibitory effect of several-fold greater potency than that of the crude venom. Fractions eluted at 18.5 min (25.4% acetonitrile concentration) and onward showed a proaggregatory but insignificant effect. It is suggested that although the venom contains pro aggregatory components, inhibition of platelet aggregation seems to be its predominant activity.

Prediction of possible sites for posttranslational modifications in human gamma crystallins: Effect of glycation on the structure of human gamma‐b‐crystallin as analyzed by molecular modeling

Crystallins are recognized as one of the long‐lived proteins of lens tissue that might serve as the target for several posttranslational modifications leading to cataract development. We have studied several such sites present in the human γ‐crystallins based either on PROSITE pattern search results or earlier experimental evidences. Their probabilities were examined on the basis of the database analysis of the γ‐crystallin sequences and on their specific locations in the constructed homology models. An N‐glycosylation site in human γD‐crystallin and several phosphorylation sites in all four human γ‐crystallins were predicted by the PROSITE search. Some of these sites were found to be strongly conserved in the γ‐crystallin sequences from different sources. An extensive analysis of these sites was performed to predict their probabilities as potential sites for protein modifications. Glycation studies were performed separately by attaching sugars to the human γB‐crystallin model, and the effect of binding was analyzed. The studies showed that the major effect of αD‐glucose (αD‐G) and αD‐glucose‐6‐phosphate (αD‐G6P) binding was the disruption of charges not only at the surface but also within the molecule. Only a minor alteration in the distances of sulfhydryl groups of cysteines and on their positions in the three‐dimensional models were observed, leading us to assume that glycation alone is not responsible for intra‐ and intermolecular disulfide bond formation. Proteins 2003.

Sea snake (Microcephalophis gracilis) hemoglobin: Primary structure and relationships to other forms

The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the α and β chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an α chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the α chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at α/β contact areas and 7 at heme contacts are exchanged in relation to the human α and β chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.

Low-Resolution Molecular Structures of Isolated Functional Units from Arthropodan and Molluscan Hemocyanin

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.