Histology of gonads and cephalic brood pouch of the marine gastropod Planaxis sulcatus of Karachi Coast (Pakistan)

This histological study of 141 specimens of adult Planaxis sulcatus from Karachi showed thepresence of only female gametes in gonadal follicles,supporting the conclusion of earlier workers that thespecies may be parthenogenetic. The gonadal folliclesof several snails were occupied by an unidentifiedtrematode parasite.     

Differential binding of tetracyclines with serum albumin and induced structural alterations in drug-bound protein

Interaction of tetracycline (TC) derivatives viz. oxytetracycline, doxycycline, demeclocycline and chlorotetracycline with bovine serum albumin (BSA) and concomitant changes in protein conformation were studied using fluorescence quenching and circular dichroism measurements. Fluorescence data revealed the presence of one to three binding sites on BSA for different TC derivatives. Binding studies with the marker ligands, warfarin and bilirubin, elucidated site-I as a primary binding site for TCs on albumin. Scatchard analysis revealed the binding affinity (K_a) and capacity (n) for these derivatives vary in the range from 0.8 to 3.2×10⁶ l/mole and 1.3–3.4, respectively. Significant reduction (60–45%) in secondary structure (-helical content) of BSA was noticed upon interaction with different TC derivatives in presence of Cu (II) ions. High affinity binding of TCs with BSA signifies drug stability. However, excessive binding at higher TC concentrations in combination with Cu (II) induces conformational change in protein structure, which may exert detrimental effect on cellular protein.     

Influence of fluoro, chloro and alkyl alcohols on the folding pathway of human serum albumin

Urea-induced equilibrium unfolding of human serum albumin (HSA) when studied by mean residue ellipticity at 222 nm (MRE(222)) or intrinsic fluorescence measurements showed a two-step, three-state transition with a stable intermediate around 4.6-5.2 M urea. The presence of 2,2,2-trifluoroethanol (TFE) resulted in a single-step, two-state transition with a significant shift towards higher urea concentration, suggesting the stabilizing effect of TFE. The free energy of stabilization (DeltaDeltaG(D)(H(2)O)) in the presence of 3.0 M TFE was determined to be 2.68 and 2.72 kcal/mol by MRE(222) and fluorescence measurements, respectively. The stabilizing potential of other alcohols on the refolding behavior of HSA at 5.0 M urea (where the intermediate exists) as studied by MRE(222) and intrinsic fluorescence measurements showed the following order: 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) > TFE > 2-chloroethanol > tert-butanol > iso-propanol > ethanol > methanol. Further, the extent of refolding at the highest concentration of alcohol was similar in all cases. The stabilizing effect of TFE on guanidine hydrochloride (GdnHCl)-induced unfolding of HSA was nearly equal to that found for urea denaturation, as reflected in the DeltaDeltaG(D)(H(2)O) value (2.38 kcal/mol). Taken together, these results suggest that the stabilizing effect of TFE and other alcohols on urea/GdnHCl-induced unfolding of HSA is higher for alcohols that contain bulky groups or fluorine atoms.     

Role of salt bridge(s) in the binding and photoconversion of bilirubin bound to high affinity site on human serum albumin

The role of salt bridge(s) (between epsilon-NH(2) groups of lysine residues of human serum albumin (HSA) and carboxyl groups of bilirubin) in the binding and photoconversion of bilirubin bound to high affinity site on HSA was investigated by covalent modification of approximately 20% internal (buried) lysine residues of HSA with acetic anhydride, succinic anhydride and O-methylisourea and white light irradiation of their complexes with bilirubin. The different HSA derivatives, namely, acetylated HSA (aHSA), succinylated HSA (sHSA) and guanidinated HSA (gHSA), thus obtained, were found to be homogeneous with respect to charge and size and characterized in detail in terms of mean residue ellipticity, Stokes radius, tryptophan fluorescence, bilirubin binding and the photochemistry of their complexes with bilirubin. All the three derivatives retained helical contents and molecular size (Stokes radius) similar to HSA except for sHSA which showed a slight increase in the Stokes radius from 3.56 to 3.64 nm. Further, fluorescence properties of aHSA and sHSA were also found to be different from HSA and gHSA. Based on difference spectral change, fluorescence quenching and fluorescence enhancement results of bilirubin bound to HSA and its derivatives, nearly 46 and 48% reduction in bilirubin binding was observed in the case of aHSA and sHSA, respectively. Both aHSA and sHSA showed a decrease of 8- and 10-fold, respectively, in association constant compared to native HSA. Although the bisignate circular dichroism (CD) spectra of an equimolar (1:1) bilirubin-HSA complex was retained by all three HSA derivatives, the intensity of both positive and negative CD Cotton effects decreased significantly in both aHSA and sHSA. gHSA which retained net charge identical to native HSA, showed little decrease in bilirubin binding and the intensity of bisignate CD Cotton effects. The photochemical reaction of bilirubin bound to aHSA and sHSA produced opposite results to those observed with HSA and gHSA. A brief (2 min) irradiation of an equimolar complex of bilirubin with both aHSA and sHSA accompanied a rapid shift (14-15 nm) in the absorption spectrum of the bound pigment towards the blue region and almost complete elimination of negative CD Cotton effects while only moderately affecting the magnitude of positive CD Cotton effects. On the other hand, similar treatment of the complexes of bilirubin with HSA and gHSA did not show any change in the absorption spectrum, only a slight decrease in the intensity of both positive and negative CD Cotton effects was observed. The fluorescence intensity of bilirubin bound to HSA and gHSA was increased upon irradiation with white light and after 30 min it was nearly twice the value observed at 0 min irradiation. Interestingly, no change in the fluorescence intensity of bilirubin bound either to aHSA or sHSA was observed upon irradiation, even on increasing the duration of irradiation to 1 h. Taken together, the results on fluorescence quenching, fluorescence enhancement, CD spectral changes and visible absorption spectroscopy suggest that salt bridge(s) of the type (-COO(-).(+)H(3)N-) in which the epsilon-NH(2) group(s) contributed by lysine residues, are not only involved in the enantioselective binding of bilirubin but also in the stereospecific photoisomerization of bilirubin bound to a high affinity site on HSA.     

Anion-induced stabilization of human serum albumin prevents the formation of intermediate during urea denaturation

The unfolding of human serum albumin (HSA), a multidomain protein, by urea was followed by far-UV circular dichroism (CD), intrinsic fluorescence, and ANS fluorescence measurements. The urea-induced transition, which otherwise was a two-step process with a stable intermediate at around 4.8 M urea concentration as monitored by far-UV CD and intrinsic fluorescence, underwent a single-step cooperative transition in the presence of 1.0 M KCl. The free energy of stabilization (DeltaDelta G(H2O)D) in the presence of 1 M KCl was found to be 1,090 and 1,200 cal/mol as determined by CD and fluorescence, respectively.The salt stabilization occurred in the first transition (0-5.0 M urea), which corresponded to the formation of intermediate (I) state from the native (N) state, whereas the second transition, corresponding to the unfolding of I state to denatured (D) state, remained unaffected. Urea denaturation of HSA as monitored by tryptophan fluorescence of the lone tryptophan residue (Trp(214)) residing in domain II of the protein, followed a single-step transition suggesting that domain(s) I and/or III is (are) involved in the intermediate formation. This was also confirmed by the acrylamide quenching of tryptophan fluorescence at 5 M urea, which exhibited little change in the value of Stern-Volmer constant. ANS fluorescence data also showed single-step transition reflecting the absence of accumulation of hydrophobic patches. The stabilizing potential of various salts studied by far-UV CD and intrinsic fluorescence was found to follow the order: NaClO(4) > NaSCN >Na(2)SO(4) >KBr >KCl >KF. A comparison of the effects of various potassium salts revealed that anions were chiefly responsible in stabilizing HSA. The above series was found similar to the electroselectivity series of anions towards the anion-exchange resins and reverse of the Hofmeister series, suggesting that preferential binding of anions to HSA rather than hydration, was primarily responsible for stabilization. Further, single-step transition observed with GdnHCl can be ascribed to its ionic character as the free energy change associated with urea denaturation in the presence of 1.0 M KCl (5,980 cal/mol) was similar to that obtained with GdnHCl (5,870 cal/mol).     

Species selectivity in poxviral complement regulators is dictated by the charge reversal in the central complement control protein modules

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.     

Viral complement regulators: the expert mimicking swindlers

The complement system is a principal bastion of innate immunity designed to combat a myriad of existing as well as newly emerging pathogens. Since viruses are obligatory intracellular parasites, they are continuously exposed to host complement assault and, therefore, have imbibed various strategies to subvert it. One of them is molecular mimicry of the host complement regulators. Large DNA viruses such as pox and herpesviruses encode proteins that are structurally and functionally similar to human regulators of complement activation (RCA), a family of proteins that regulate complement. In this review, we have presented the structural and functional aspects of virally encoded RCA homologs (vRCA), in particular two highly studied vRCAs, vaccinia virus complement control protein (VCP) and Kaposi's sarcoma-associated herpesvirus complement regulator (kaposica). Importance of these evasion molecules in viral pathogenesis and their role beyond complement regulation are also discussed.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Effect of lysine modification on the conformation and indomethacin binding properties of human serum albumin.

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in alpha-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.