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GTP cyclohydrolase II (GCHII) is one of the rate limiting enzymes in riboflavin biosynthesis pathway and is shown to be a potential drug target for most of the pathogens. Previous biochemical and structural studies have identified the active site residues and elucidated the steps involved in the catalytic mechanism of GCHII. However, the last ∼20–25 C-terminal residues of GCHII remains unstructured in all the crystal structures determined to date and their role in the catalytic activity, if any, remains elusive. Therefore, to understand the role of these unstructured C-terminal residues, a series of C-terminal deletion mutants of GCHII from Helicobacter pylori (hGCHII) were generated and their catalytic activity was compared with its wild-type. Surprisingly, none of the C-terminal deletion mutants shows any enzymatic activity indicating that these are essential for GCHII function. To get additional insights for such loss of activity, homology models of full-length and deletion mutants of hGCHII in complex with GTP, Mg2+, and Zn2+ were generated and subjected to molecular dynamics simulation studies. The simulation studies show that a conserved histidine at 190th position from the unstructured C-terminal region of hGCHII interacts with α-phosphate of GTP. We propose that His-190 may play a role in the hydrolysis of pyrophosphate from GTP and in releasing the product, DARP. In summary, we demonstrate that the unstructured C-terminal residues of GCHII are important for its enzymatic activity and must be considered during rational drug designing. Communicated by Ramaswamy H. Sarma

The unintended crystallization of proteins which generally originate from the expression host instead of the target recombinant proteins is periodically reported. Despite the massive technological advances in the field, assigning a structural model to the corresponding diffraction data is not a trivial task. Here, the structure of acyl-carrier protein synthase (AcpS) from Mycobacterium smegmatis (msAcpS), which crystallized inadvertently in an experimental setup to grow crystals of a Mycobacterium tuberculosis protein using M. smegmatis as an expression system, is reported. After numerous unsuccessful attempts to solve the structure of the target protein by the molecular-replacement method no convincing solutions were obtained, indicating that the diffraction data may correspond to a crystal of an artifactual protein, which was finally identified by the Sequence-Independent Molecular replacement Based on Available Databases (SIMBAD) server. The msAcpS structure was solved at 2.27 Å resolution and structural analysis showed an overall conserved fold. msAcpS formed a trimeric structure similar to those of other reported structures of AcpS from various organisms; however, the residues involved in trimer formation are not strictly conserved. An unrelated metal ion (Ni2+), which was possibly incorporated during protein purification, was observed in the proximity of His49 and His116. Structural and sequence differences were observed in the loop connecting the α3 and α4 helices that is responsible for the open and closed conformations of the enzyme. Moreover, the structural analysis of msAcpS augments the current understanding of this enzyme, which plays a crucial role in the functional activation of acyl-carrier proteins in the fatty-acid biosynthesis pathway.

Coiled coil domain-nucleotide binding site-leucine rich repeat (CC-NBS-LRR; CNL) proteins are highly conserved family of plant disease resistance proteins, remarkably comprise of coiled-coil domain, which plays significant role in plant innate immunity. The present study reports that moderately elicited oligomerization of plant CNL like protein fragment (CNL-LPF) in presence of ATP/Mg using various biophysical methods Circular dichroism (CD) results depicted a substantial increase in β-sheet structure content of CNL-LPF. ATP/Mg induced conformational change in protein was observed by increase in blue shift with extrinsic fluorescence measurement, which indicates the exposure of hydrophobic regions of CNL-LPF and leads to self-association i.e. oligomerization. Likewise, cluster of protein oligomer and alteration in protein surface morphology were observed in presence of ATP/Mg by Transmission electron microscopy (TEM) and Atomic force microscopy (AFM), respectively. Also, augmented antiproliferation of HT1376 cells (urinary bladder cancer cell lines) was observed by CNL-LPF in presence of ATP/Mg. In conclusion, the current study illustrates that extent of CNL-LPF oligomerization was enhanced in presence of ATP/Mg (as compared to its absence). Utilization of enhanced oligomerization property of CNL-LPF as an anti-proliferative agent needs more assessment.

Background: A well-known tissue marker of ovarian cancer, Human Epididymis protein 4 (HE4) is the member of whey acidic four-disulfide core proteins family. Purified from human seminal plasma and characterized as a cross-class protease inhibitor, HE4 was proposed to shield spermatozoa against proteolytic factors. However, its exact biological function is unknown. Proteins usually function in conjunction with other proteins in the system and thus, identification and analysis of protein networks become essential to decode protein functions. Objective: This study was performed to explore possible role(s) of HE4 in reproductive physiology via identification of its interactome in human seminal plasma. Methods: HE4 binding proteins were identified through co-immunoprecipitation and MALDITOF/ MS analysis. Also, HE4 was quantified by ELISA in fertile and infertile human seminal plasma samples. Results: Ten HE4 binding proteins were identified, viz. protein phosphatase 1 regulatory subunit 21, protein kinase CLK3, Ankyrin repeat domain-containing protein36A, prostatic acid phosphatase, KIF5C, Spectrin repeat containing, nuclear envelope 1, isoform CRAf, tropomyosin 4, vezatin, utrophin and fibronectin1. This interaction network suggests that HE4 plays multiple roles, specifically in capacitation, sperm motility and maturation. Further, HE4 concentration in human seminal plasma samples was determined by Elisa. Higher HE4 expression in normozoospermia compared to azoospermia and asthenozoospermia affirms its importance in fertilization. Conclusion: Based on identified interactome, it is plausible that HE4 plays a crucial role in fertilization, specifically in sperm maturation, motility and capacitation.

The current treatment therapies available for malignant gliomas are inadequate. There is an urgent need to develop more effective therapies by characterizing the molecular pathogenesis of the disease. Over expression of platelet-derived growth factor (PDGF) ligands and receptors have been reported in malignant gliomas. Platelet-derived growth factor associated protein-1 (PDAP-1) is reported to modulate the mitogenic activity of PDGF ligands, but to date, there is no information concerning its role in PDGF-mediated glioma cell proliferation. This study aimed to characterize the role of PDAP-1 in PDGF-mediated glioma proliferation. The expression of PDAP-1 was observed to be significantly increased (p

Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample.

GTP cyclohydrolase II (GCHII), catalyzes the conversion of GTP to 2,5-diamino-6-β-ribosyl-4(3H)-pyrimidinone-5'-phosphate and has been shown to be essential for pathogens. Here we describe the biochemical, kinetic and structural characterization of GCHII from Helicobacter pylori (hGCHII). The crystal structure of hGCHII, unlike other GCHII structures, revealed that cysteines at the active site existed in oxidized state forming two disulfide bonds and lacked Zn(2+) that was shown to be indispensable for catalytic activity in other species. However, incubation of hGCHII with hydrogen peroxide, an oxidizing agent, followed by PAR-assay showed that Zn(2+) was intrinsically present, indicating that all cysteines at the catalytic site remained in reduced state. Moreover, site directed mutagenesis of catalytic site cysteines revealed that only three, out of four cysteines were essential for hGCHII activity. These results, though, indicated that hGCHII crystallized in oxidized form, the expulsion of Zn(2+) upon oxidation of catalytic cysteines revealed its ability to act in response to the redox environment. Exploring further, incubation of hGCHII with reversible thiol modifying agent S-methyl-methane-thiosulfonate resulted in loss of GCHII activity due to oxidation of its cysteine residues as revealed by mass spectrometry studies. However, addition of reducing agent DTT partially restored the hGCHII catalytic activity. Taken together, these results demonstrate that hGCHII can regulate its catalytic activity depending on the redox environment, a function hitherto unknown for GCHII.

High-temperature requirement A (HtrA) proteins, which are members of the heat-shock-induced serine protease family, are involved in extracytoplasmic protein quality control and bacterial survival strategies under stress conditions, and are associated with the virulence of several pathogens; they are therefore major drug targets. Mycobacterium tuberculosis possesses three putative HtrAs: HtrA1 (Rv1223), HtrA2 (Rv0983) and HtrA3 (Rv0125). Each has a cytoplasmic region, a transmembrane helix and a periplasmic region. Here, the crystal structure of the periplasmic region consisting of a protease domain (PD) and a PDZ domain from an M. tuberculosis HtrA1 mutant (mHtrA1S387A) is reported at 2.7 Å resolution. Although the mHtrA1S387A PD shows structural features similar to those of other HtrAs, its loops, particularly L3 and LA, display different conformations. Loop L3 communicates between the PDs of the trimer and the PDZ domains and undergoes a transition from an active to an inactive conformation, as reported for an equivalent HtrA (DegS). Loop LA, which is responsible for higher oligomer formation owing to its length (50 amino acids) in DegP, is very short in mHtrA1S387A (five amino acids), as in mHtrA2 (also five amino acids), and therefore lacks essential interactions for the formation of higher oligomers. Notably, a well ordered loop known as the insertion clamp in the PDZ domain interacts with the protease domain of the adjacent molecule, which possibly aids in the stabilization of a trimeric functional unit of this enzyme. The three-dimensional structure of mHtrA1S387A presented here will be useful in the design of enzyme-specific antituberculosis inhibitors.

Ovarian cancer (OC) is one of the most lethal cancers among all gynecological malignancies. An effective and non-invasive screening approach is needed urgently to reduce high mortality rate. The purpose of this study was to identify the salivary protein signatures (SPS) for non-invasive detection of ovarian cancer. Differentially expressed SPS were identified by fluorescence-based 2D-DIGE coupled with MALDI/TOF-MS. The expression levels of three differential proteins (Lipocalin-2, indoleamine-2, 3-dioxygenase1 (IDO1) and S100A8) were validated using western blotting and ELISA. Immunohistochemistry and qRT-PCR were performed in an independent cohort of ovarian tumor tissues. 25 over expressed and 19 under expressed (p

Background Early Childhood Caries (ECC) is most common chronic infectious disease of childhood. Diagnosis of dental caries has been limited to clinical, visual and radiographic methods but its inflammatory component remained unexplored. Hence, this study aims to evaluate salivary levels of inflammatory cytokines in children with ECC to assess their potential as non-invasive biomarkers. Methods 50 subjects were recruited (25 ECC patients and 25 healthy children). Saliva samples were taken from all subjects and collected again from patients after rehabilitative intervention. Levels of IL-6, IL-8 and TNF- α were determined using ELISA. Cytokines level were statistically correlated with each other and with DMF score along with ROC curve analysis. Results Salivary levels of IL-6, IL-8 & TNF-α were significantly higher in patients which got significantly reduced after rehabilitative intervention. Levels of these cytokines were significantly associated with severity of dental caries. These cytokines were correlating with each other along with DMF score upon Spearman correlation. ROC curve reveals optimum sensitivity and specificity of these cytokines for diagnosis in ECC with absolute levels observed for IL-6. Conclusions Significant elevation of IL-6, IL-8 and TNF-α with optimum sensitivity and specificity might imply their involvement as potential non-invasive diagnostic/prognostic markers in ECC.

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.

Background The p38α MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-α and IL-1β. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38α. Methods A tetrapeptide, VWCS as p38α inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38α was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry. Results The percentage inhibition of designed VWCS against pure p38α protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the KD and IC50 of the selective peptide as 7.22 × 10− 9 M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC50 value of 10 μM and induced apoptosis by activating Caspase 3 and 7. Conclusions VWCS efficiently interacted at the ATP binding pocket of p38α with high potency and can be used as a potent inhibitor in case of HNSCC. General significance VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38α MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.

Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

Identification of protein–protein interactions is vital for complete understanding of a biological process and for functional characterization of a protein in related biochemical pathways. In this study, we performed analysis of prolactin inducible protein (PIP) interactions in human seminal plasma. PIP and its interacting partners were co-immunoprecipitated, analyzed by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Three major interacting partners were identified, viz. human serum albumin, zinc-α-2 glycoprotein and semenogelin I fragments. This is the first report of interaction between PIP and semenogelin I fragments in human seminal plasma or elsewhere with a suggestive role in reproductive physiology which might be helpful for spermatozoa to acquire their motility.

Scarcely understood defects lead to asthenozoospermia, which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma data set, which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the data set.It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples. This is despite the results that some of the seminal plasma proteins have big fold changes among classes but they fall short of statistical significance. S-Plot of the sperm proteomic data set generated some high confidence targets, which might be implicated in sperm motility pathways. These proteins also had the area under the curve value of 0.9 or 1 in ROC curve analysis.Various pathways were either enriched in these proteomic data sets by pathway analysis or they were searched by their constituent proteins. Some of these pathways were axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly among others. The mass spectrometric data is available via ProteomeXchange with identifier PXD004098.

CC-NBS-LRR (CNL) plant proteins are related with highly conserved family of disease resistance protein distinguished by a coiled-coil domain, which plays an important role in innate immunity. The present study reports the purification and identification of CNL like protein fragment (CNL-LPF) by two step chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/MS), respectively. Furthermore, current study also illustrated the development of polyclonal antibody against purified CNL-LPF, which was used for immunolocalization of CNL-LPF in cytoplasm of cotyledon, using Fluorescence microscopy and Transmission electron microscopy. Lastly, present study also demonstrates in vitro oligomerization of purified CNL-LPF with multiple bands on 4-10 % gradient native-PAGE; each band representing a small fraction of each oligomer population as evident by immunoblots. In conclusion, the current study deals with the purification and polyclonal antibody development against CNL-LPF.

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dose-dependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.

Background: Prolactin inducible protein (PIP) is a ~17 kDa protein, which is known to play vital roles in immunoregulation, fertility, antimicrobial activity, apoptosis and tumour progression.Objectives: This study reports quantification of PIP concentration in human seminal plasma (SP) samples.Methodology: PIP was purified by immunoprecipitation and its concentration in human SP samples was quantified by ELISA method.Results: Average concentration of PIP in normozoospermia, oligozoospermia and azoospermia was 290.3 ± 71.5 µg/mL, 306.4 ± 71.2 µg/mL and 60.5 ± 23.6 µg/mL respectively.Conclusion: There was no significant variation in PIP levels in normozoospermia and oligozoospermia while its expression was down-regulated in azoospermia, indicating that PIP may be a plausible marker of azoospermia.

Pancreatic cancer is very difficult to diagnose in its early stage. Molecular marker and imaging have not proven to be accurate modalities for screening of pancreatic cancer. This study aims to develop p38β as a protein marker for pancreatic cancer and to design peptide inhibitor against the same. The serum p38β level of pancreatic cancer (n = 35; 5.06 μg/mL) was twofold higher compared to that of the chronic pancreatitis (n = 10; 2.92 μg/mL) and matched normal control (n = 10; 2.86 μg/ml) (p

Glycoproteins have immense clinical importance and comparative glycoproteomics has become a powerful tool for biomarker discovery and disease diagnosis. Seminal plasma glycoproteins participate in fertility related processes including sperm-egg recognition, modulation of capacitation and acrosome reaction inhibition. Affinity chromatography using broad specificity lectin such as Con A is widely applied for glycoproteins enrichment. More notably, Con A-interacting fraction of human seminal plasma has decapacitating activity which makes this fraction critically important. In our previous study, we isolated Con A-interacting glycoproteins from human seminal plasma and subsequently identified them by mass spectrometry. Here, we report the computational analysis of these proteins using bioinformatics tools. The analysis includes: prediction of glycosylation sites using sequence information (NetNGlyc 1.0), functional annotations to cluster these proteins into various functional groups (InterProScan and Blast2GO) and identification of protein interaction networks (STRING database). The results indicate that these proteins are involved in various biological processes including transport, morphogenesis, metabolic processes, cell differentiation and homeostasis. The clusters illustrate two major molecular functions - hydrolase activity (6) and protein (4)/carbohydrate (1)/lipid binding (1). The large interactomes of proteins point towards their versatile roles in wide range of biological processes.

The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.

Keyhole limpet hemocyanin (KLH) is widely used as an immune stimulant and hapten carrier derived from a marine mollusc Megathura crenulata. To provide details of the stability and equilibrium of KLH, different intermediate species were investigated with a series of biophysical techniques: circular dichroism, binding of hydrophobic dye, 1-anilino-8-naphthalene sulfonic acid, acrylamide-induced fluorescence quenching, thermal stability and dynamic light scattering. KLH in its native state at pH 7.4 exists in the stable didecameric form with hydrodynamic radii (R h) of 28.22 nm, which is approximately equal to a molecular mass of 8.8 ± 0.6 MDa. The experimental results demonstrated the presence of two structurally distinct species in the conformational transition of KLH under acidic conditions. One species populates at pH 2.8, characterized as decameric (4.8 ± 0.2 MDa; R h = 22.02 nm), molten globule-like state, while the other accumulates at pH 1.2 and is characterized as a tetramer (2.4 ± 0.8 MDa; R h = 16.47 nm) with more organized secondary and tertiary structures. Our experimental manipulation of the oligomeric states of KLH has provided data that correlate well with the known oligomeric forms obtained from total KLH formed in vivo and extends our understanding of multimer formation by KLH. The results are of particular interest in light of the important role of the mechanistic pathway of pH-dependent structural changes of Hc stability in the biochemical and medical applications of these respiratory proteins.

Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identifi ed these proteins. Forty different types of proteins were identified. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identified 8% of proteins with unknown functions, although their expression in seminal fluid has been documented. Proteins of seminal fluid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future.

Human amniotic fluid is of both maternal and fetal origin; it protects the fetus and provides the environment for growth and development of the fetus. We used a proteomics-based approach for targeting and purifying human phosphate binding protein, a member of the DING family of proteins from amniotic fluid, using Blue Sepharose CL-6B, DEAE-Sephacel and gel filtration chroma- tography. The protein had earlier been reported to be serendipitously purified along with PON1 (paraoxonase 1). It was identified using electro-spray-ionization-time-of-flight mass spectrometry and was found to be human phosphate binding protein. Human phosphate binding proteins have been reported to play a role as phosphate scav- engers and may have a protective function against phosphate-related disorders, such as atherosclerosis, diabetes and kidney stones.

Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield.

Zinc alpha2-glycoprotein (ZAG) is present at high concentration in human seminal plasma, and considered as soluble homologue of MHC-I. ZAG is a well-known biomarker for prostate and breast carcinomas. We have purified a naturally occurring complex of ZAG with Prolactin inducible protein (PIP), which is also a well-known biomarker for the same. The ZAG-PIP complex has been isolated and purified by simple chromatographic techniques in a reproducible two-step process, using ion exchange and gel-permeation chromatography and subsequently identified by MS. The complex between ZAG and PIP is formed by non-covalent interactions. The purity and molecular mass was determined by SDS-PAGE, which shows the bands corresponding to 40 kDa and 14 kDa, which were also confirmed by MALDI-TOF. Dynamic light scattering (DLS) experiments also showed hydrodynamic radii corresponding to 54, 40 and 14 kDa for ZAG-PIP complex, ZAG and PIP respectively. The concentration dependent aggregation of this complex has also been observed. Fluorescence analysis reveals that complexes have similar binding affinities as native ZAG, for their proposed ligands like arachidonic acid, polyethylene glycol and synthetic peptide. This is the first report on purification and characterization of a naturally occurring complex of ZAG-PIP in human seminal fluid.

Zinc α2-glycoprotein (ZAG) is a protein of interest because of its ability to play many important functions in the human body, including fertilization and lipid mobilization. After the discovery of this molecule, during the last 5 decades, various studies have been documented on its structure and functions, but still, it is considered as a protein with an unknown function. Its expression is regulated by glucocorticoids. Due to its high sequence homology with lipid-mobilizing factor and high expression in cancer cachexia, it is considered as a novel adipokine. On the other hand, structural organization and fold is similar to MHC class I antigen-presenting molecule; hence, ZAG may have a role in the expression of the immune response. The function of ZAG under physiologic and cancerous conditions remains mysterious but is considered as a tumor biomarker for various carcinomas. There are several unrelated functions that are attributed to ZAG, such as RNase activity, regulation of melanin production, hindering tumor proliferation, and transport of nephritic by-products. This article deals with the discussion of the major aspects of ZAG from its gene structure to function and metabolism. (Mol Cancer Res 2008;6(6):892–906)

Prostate specific antigen (PSA) is a member of kallikrein family having serine protease-like activity and acts as a prognostic marker of prostate carcinoma. Various studies have been performed on inhibition of PSA and such targeting requires the identification of highly selective peptide inhibitors. PSA was purified from human seminal plasma by rapid and efficient methods, and binding studies for various peptides were carried out by fluorescence spectroscopy and SPR. The ‘S’ of PSA is predominated by hydrophobic residues, and hence many hydrophobic peptides were used to determine their binding affinity to PSA by fluorescence spectroscopy. We observed that LLFW, FFKW, and KFW binds strongly to PSA, among them LLFW showed strong binding. SPR also showed strong binding affinity of PSA toward peptides with hydrophobic and basic residues. Among the peptides used, FWYS showed dramatic increase in binding affinity (10−10M). The peptides analyzed for binding studies, suggests that peptide with Trp residue along with basic or hydrophobic amino acids may be useful for designing specific inhibitors for PSA. The strong affinities of designed peptides for PSA can be a valuable tool for designing therapeutic agents for prostate carcinomas. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of β-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.

Disintegrins constitute a family of potent polypeptide inhibitors of integrins. Integrins are transmembrane heterodimeric molecules involved in cell-cell and cell-extracellular matrix interactions. They are involved in many diseases such as cancer and thrombosis. Thus, disintegrins have a great potential as anticancer and antithrombotic agents. A novel heterodimeric disintegrin was isolated from the venom of saw-scaled viper (Echis carinatus) and was crystallized. The crystals diffracted to 1.9 A resolution and belonged to space group P4(3)2(1)2. The data indicated the presence of a pseudosymmetry. The structure was solved by applying origin shifts to the disintegrin homodimer schistatin solved in space group I4(1)22 with similar cell dimensions. The structure refined to the final R(cryst)/R(free) factors of 0.213/0.253. The notable differences are observed between the loops, (Gln39-Asp48) containing the important Arg42-Gly43-Asp44, of the present heterodimer and schistatin. These differences are presumably due to the presence of two glycines at positions 43 and 46 that allow the molecule to adopt variable conformations. A comparative analysis of the surface-charge distributions of various disintegrins showed that the charge distribution on monomeric disintegrins occurred uniformly over the whole surface of the molecule, while in the dimeric disintegrins, the charge is distributed only on one face. Such a feature may be important in the binding of two integrins to a single dimeric disintegrin. The phylogenetic analysis developed on the basis of amino acid sequence and three-dimensional structures indicates that the protein diversification and evolution presumably took place from the medium disintegrins and both the dimeric and short disintegrins evolved from them.

Ribosome-inactivating proteins (RIPs) are toxins involved in plant defense. How the plant prevents autotoxicity is not yet fully understood. The present study is the first structural evidence of a naturally inhibited form of RIP from a plant. Himalayan mistletoe RIP (HmRIP) was purified from Viscum album leaves and crystallized with lactose. The structure was determined by the molecular replacement method and refined at 2.8-A resolution. The crystal structure revealed the presence of high quality non-protein electron density at the active site, into which a pteridine derivative (2-amino 4-isopropyl 6-carboxyl pteridine) was modeled. The carboxyl group of the ligand binds strongly with the key active site residue Arg(162), nullifies the positive charge required for catalysis, and thereby acts as a natural inhibitor. Lectin subunits of RIPs have two active sugar-binding sites present in 1alpha- and 2gamma-subdomains. A third functionally active site has been identified in the 1beta-subdomain of HmRIP. The 1beta-site is active despite the absence of conserved polar sugar-binding residues. Loss of these residues is compensated by the following: (i) the presence of an extended site where the penultimate sugar also interacts with the protein; (ii) the interactions of galactose with the protein main chain carbonyl and amide nitrogen atoms; (iii) the presence of a well defined pocket encircled by four walls; and (iv) a favorable stacking of the galactose ring with Tyr(66) besides the conserved Phe(75). The mode of sugar binding is also distinct at the 1alpha and 2gamma sugar-binding sites.

An essential protein for bacterial growth, GTPase-Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti-sigma-F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above-mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg-Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg-Usfx interaction. Surface plasmon resonance results clearly suggest that N-terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.

Lagenaria siceraria (bottle gourd) class belongs to Magnoliopsida family curcurbitaceae that is a traditionally used medicinal plant. Fruit of this plant are widely used as a therapeutic vegetable in various diseases, all over the Asia and Africa. Various parts of this plant like fruit, seed, leaf and root are used as alternative medicine. In the present study, primarily, we have focused on proteomic analysis of L. siceraria seed using phenol extraction method for protein isolation. Twenty-four colloidal coomassie blue stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) after resolving on two-dimensional gel electrophoresis. Out of 24 identified protein spots, four were grouped as unidentified proteins which clearly suggest that less work has been done in the direction of plant seed proteomics. These proteins have been found to implicate in various functions such as biosynthesis of plant cell wall polysaccharides and glycoproteins, serine/threonine kinase activity, plant disease resistance and transferase activity against insects by means of insecticidal and larval growth inhibitory, anti-HIV, antihelmintic and antimicrobial properties. By Blast2GO annotation analysis, amongst the identified proteins of L. siceraria, molecular function for majority of proteins has indispensable role in catalytic activity, few in binding activity and antioxidant activity; it is mostly distributed in cell, organelle, membrane and macromolecular complex. Most of them involved in biological process such as metabolic process, cellular process, response to stimulus, single organism process, signalling, biological recognition, cellular component organization or biogenesis and localization.

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity.

Protease and fatty acids are known to serve as pathogenic factors against root-knot nematodes. Here, we utilized Karanja deoiled cake as a nitrogen source for the first time in induction of protease by Paecilomyces lilacinus. Fatty acids, especially butyric acid, have also been detected in the same fungal culture filtrate.

Syzygium cumini is traditionally used medicinal plant. The different part of the plant such as bark, leaves, seed and fruits are widely used as an alternative medicine in various diseases. Although the scientific community has a strong interest on S. cumini seed biochemistry focusing on metabolite composition, proteins have not yet been investigated. In the present study, we have applied a proteomic approach to study the proteome of the S. cumini seed using phenol extraction method for protein isolation, which were never analysed before. Fifteen brightly silver stained protein spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after resolving on two-dimensional gel electrophoresis. These proteins have been found to involve in various functions such as antifungal, sulphur metabolism, carbohydrate metabolism, fruit ripening and softening, dormancy breaking and seed germination, hormone signalling, secondary metabolite transport, defence and stress response, nitrogen metabolism, synthesis and stabilization. Amongst the identified protein, lactoferrin was a mammalian origin protein with high nutritious and pharmaceutical value, which was purified by different types of chromatographic techniques and confirmed by western blotting. The antibacterial activity of lactoferrin was assessed by disc diffusion assay. We suggest that the protein constituents of S. cumini may have role in various functions required for plant physiology and its dietary values.

Carboxypeptidase E (CPE) cleaves basic amino acid residues at the C-terminal end and involves in the biosynthesis of numerous peptide hormones and neurotransmitters. It was purified from human seminal plasma by ion exchange, heparin affinity and gel filtration chromatography followed by identification through SDS-PAGE and MALDI-TOF/MS analysis, which was further confirmed by western blotting. CPE was characterized as glycoprotein by Periodic Acid Schiff (PAS) staining and treating with deglycosylating enzyme N-glycosidase F. The interaction of CPE with heparin was illustrated by surface plasmon resonance (SPR) and in silico interaction analysis. The association constant (KA) and dissociation constant (KD) of CPE with heparin was determined by SPR and found to be 1.06 × 105 M and 9.46 × 10−6 M, respectively. It was detected in human spermatozoa also by western blotting using mouse anti-CPE primary antibody. 20–100 μg/ml concentration of CPE was observed as highly effective in killing Escherichia coli by colony forming unit (CFU) assay. We suggest that CPE might act not only in the innate immunity of male reproductive tract but also regulate sperm fertilization process by interacting heparin.

Glycosaminoglycans (GAGs) have crucial roles in cell-cell interaction and communication. The communication between sperm and egg during fertilization is the finest example of intercellular communication involving a protein-carbohydrate recognition system. GAGs, especially heparin, are implicated in various processes, such as capacitation, acrosome reaction (AR), and sperm nuclei decondensation by interacting with a wide range of proteins, leading to fertilization. Seminal plasma (SP) comprises of multiple proteins that bind to heparin and related GAGs. Heparin binding proteins (HBPs) originating from secretions of the male accessory sex glands are known to play a vital role during fertilization events. They interact with GAGs present in the female genital tract and enhance the subsequent zona pellucida-induced AR, and thus have been correlated with fertility in some species. Several carbohydrate and zona pellucida-binding proteins, many of which belong to the spermadhesin family, are identified as HBPs. Many studies have been documented about the potential physiological role of some HBPs in various steps of fertilization. However, there is insufficient knowledge about functions executed by various HBPs and their exact mechanism and pathways. An in-depth knowledge of HBPs and their role in fertilization is of fundamental importance to resolve biological pathways and protein interactions at the molecular level. This review surveys some of the relevant findings supporting the potential role of heparin and HBPs in reproduction. It also describes consensus heparin binding sites emerging from known literature on HBPs that interact with heparin.

Epididymal proteins represent the factors necessary for maturation of sperm and play a crucial role in sperm maturation. HE-4, an epididymal protein, is a member of whey acidic protein four-disulfide core (WFDC) family with no known function. A WFDC protein has a conserved WFDC domain of 50 amino acids with eight conserved cystine residue. HE-4 is a 124 amino acid long polypeptide with two WFDC domains. Here, we show that HE-4 is secreted in the human seminal fluid as a disulfide-bonded homo-trimer and is a cross-class protease inhibitor inhibits some of the serine, aspartyl and cysteine proteases tested using hemoglobin as a substrate. Using SPR we have also observed that HE-4 shows a significant binding with all these proteases. Disulfide linkages are essential for this activity. Moreover, HE-4 is N-glycosylated and highly stable on a wide range of pH and temperature. Taken together this suggests that HE-4 is a cross-class protease inhibitor which might confer protection against microbial virulence factors of proteolytic nature.

Prolactin inducible protein (PIP) is a 17 kDa glycoprotein. It binds to many proteins including fibrinogen, actin, keratin, myosin, immunoglobulin G, CD4, and human zinc-alpha-2 glycoprotein. Its ability to bind a large array of proteins indicates its multifaceted role in various biological processes, such as fertility, immunoregulation, antimicrobial activity, apoptosis, and tumor progression. Here, we present the first report of native human serum albumin (HSA)-PIP complex formation in seminal plasma. The complex was purified by chromatographic separation techniques, analyzed by gel electrophoresis, identified by MALDI-TOF mass spectrometry and validated by co-immunoprecipitation coupled with western blotting experiments. Moreover, the behavior of complex in solution was analyzed by dynamic light scattering and interacting residues were identified by in silico protein-protein docking. The purified protein complex shows two bands (67 kDa and 17 kDa) on SDS-PAGE gel and a single band (~85 kDa) on native PAGE gel. The predicted complex structure has 13 intermolecular hydrogen bonds, which may contribute to the overall stability of the complex. As HSA has been known to preserve the motility of sperm, native HSA-PIP complex formation may point towards an important role of PIP, which can directly be correlated with male fertility/infertility.

Boron is reported in this study to stabilize the structure of heme proteins exposed to peroxides. The oxidized heme protein (15 microM) was treated with H(2)O(2) (10mM) in 1M glycine-NaOH buffer (pH 9.2) at 25 degrees C in absence/presence of boron, and characterized by visible absorption spectroscopy, gel exclusion chromatography, native PAGE, HPLC and DLS. Spectral analysis of exposed heme proteins revealed a decrease in absorbance in the Soret region, which was stabilized by boron. The native PAGE analysis of exposed heme proteins showed high molecular weight products; the band intensity was lesser in presence of boron. Further, elution profile of the exposed heme proteins on Sephadex G-200 column and HPLC revealed more than one peak (aggregate formation) when compared to the respective untreated proteins. DLS, which measures the hydrodynamic radius (R(H)), was used to ascertain whether the peaks correspond to monomer, dimer or aggregate forms. The R(H) of boron pretreated heme proteins was close to R(H) of the respective heme protein. Non-heme protein RNase did not show any change when exposed to peroxide. Taken together, results conclude that boron stabilizes the structure of heme proteins, which might be due to specific sites on heme proteins that can bind to borate ions.

beta-Microseminoprotein (beta-MSP) is a small cysteine-rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino-acid sequences of beta-MSP proteins suggests that the protein is a rapidly evolving protein. The function of beta-MSP is poorly understood. Furthermore, no crystal structure has been reported of any beta-MSP; therefore, determination of the crystal structure of beta-MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X-ray diffraction analysis of beta-MSP from human seminal plasma are described. The protein was purified using anion-exchange and size-exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4(3)22 and contained three beta-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 A resolution.

Heparin binding proteins (HBPs) from human seminal plasma (HSP) were obtained by heparin affinity and size exclusion chromatography. The aggregation/disaggregation of HBPs was followed by dynamic light scattering (DLS) in presence of various physiological ligands such as CaCl(2), NaCl, EDTA, cholesterol, adenosine, D-glucose and D-fructose. The aggregation pattern of lactoferrin was also analyzed and compared. The study of interactions of HBPs of HSP may contribute to an understanding mechanisms underlying the fertilization process.

Background Prostate specific antigen (PSA) present in human seminal fluid exists in many isoforms due to different sequence, glycosylation pattern and polypeptide length. Its presence in various stages of cancer has already been reported. Methods We identified 8 forms of PSA followed by purification of 3 forms. We compared their binding affinities to designed synthetic peptides, by using surface plasmon resonance (SPR). PSA forms were purified using various chromatographic procedures and characterized by 2D gel electrophoresis (2DE), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Results The heterogeneity of purified PSA was demonstrated by 2DE analysis. The peptides were designed on the basis of cleavage map of insulin-like growth factor binding protein 3 (IGFBP-3) subsequently, these peptides showed the binding affinities in the range of 10− 5 to 10− 12 M. Out of 7 peptides, VLLH showed maximum affinity to intact PSA, while FLSYK showed maximum affinity to cleaved forms of PSA. On the other hand, FLSYK in the presence of zinc showed higher affinity to intact PSA. These peptides showed higher affinity to PSA compared to zinc, which is a known inhibitor of PSA. Conclusions This study reports purification and characterization of 3 forms of PSA simultaneously. Furthermore, we have reported specific peptides for different forms of PSA which are either responsible for prostate cancer (PCa) or benign prostatic hyperplasia (BPH). Our study aids the existing information on categorizing the molecular heterogeneity of PSA.

Based on unique biology of prostate cancer, prostate-specific antigen could be a useful target for prostate cancer therapies. Such targeting requires the identification of highly selective inhibitor-binding sites. Three-dimensional structure was calculated by homology modeling. The overall structure of human prostate-specific antigen is composed of two β-barrel domain, kallikrein loop and active-site triad His 57 , Asp 102 , and Ser 195 . Structure of human prostate-specific antigen is quite similar to hK-1 and HPK-3. The major differences were observed at kallikrein loop and position of active site. The substrate-binding pocket is predominated by hydrophobic residues and the bottom of the specificity pocket contains Ser 189 as in chymotrypsin, which provides substrate specificity. The hydrophobic, and preferentially aromatic (Trp215), amino acid residues are determinant of substrate binding due to the presence of hydrophobic crevice between Tyr 99 and Trp 215 . Crystal structure of human prostate-specific antigen is not determined till now and hence the report on an accurate and comparative model of human prostate-specific antigen is probably to help in understanding their functional network and finally could be helpful in structure-based rational drug designing.

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35CHAPSCTABSDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.

Human seminal plasma contains a large array of proteins required for the normal physiology and metabolism of spermatozoa. These are mainly secreted from prostate epithelium, testes, and seminal vesicles. Fortunately, many of these are found to be present at elevated concentration in seminal plasma and act as a biomarker of different carcinomas as their levels are also enhanced in serum and are found to be involved in tumor progression and metastasis apart from fertility. The proteins which were overexpressed in the seminal plasma of prostate carcinoma patients were identified by 2-DE and MALDI-TOF/MS. We have designed a strategy to purify these four proteins prostate specific antigen (PSA), prostatic acid phosphatase (PAP), Zinc alpha2-glycoprotein (ZAG), and progastricsin (PG), together in homogeneity by using simple chromatographic techniques. Acidic and basic fractions of human seminal plasma were separated by ion exchange chromatography and further purified by gel permeation chromatography. Our results form a new and valuable resource for those attempting structure-based drug designing for prostate and other cancers where the amount of proteins is required in plenty and in native form.