Improving the accuracy of pancreatic cancer clinical staging by exploitation of nanoparticle-blood interactions: A pilot study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) early diagnosis is  crucial  and new, cheap and user-friendly techniques for biomarker identification  are  needed. "Protein corona" (PC) is emerging a new bio-interface potentially useful in tumor early diagnosis. In a previous investigation, we showed that relevant differences between the  protein patterns of  PCs formed on lipid NPs after exposure to PDAC and non-cancer plasma  samples exist. To extend that research, We performed this pilot study to investigate the effect of PDAC tumor size and distant metastases on PC composition. METHODS: Twenty PDACs were clinically staged according to the UICC TNM staging system 8 t h Edition. Collected plasma samples were let to interact with lipid NPs; resulting PCs were characterized by SDS-PAGE. To properly evaluate changes in the PC, the protein intensity profiles were reduced to four regions of molecular weight: < 25 kDa, 25-50 kDa, 50-120 kDa, > 120 kDa.  RESULTS: Data analysis allowed to distinguish T1-T2 cases from T3 and above all from metastatic ones (p < 0.05). Discrimination power was particularly due to a subset of plasma proteins with molecular  weight comprised between 25-50 kDa  and 50-120 kDa. CONCLUSIONS: PC composition is critically influenced by tumor size and presence of distant metastases in PDAC. If our findings will be further confirmed, we envision that future developments of cheap and user-friendly PC-based tools will allow to improve the accuracy of PDAC clinical staging, identifying among resectable  PDACs with potentially better prognosis (i.e. T1 and T2) those at higher risk of occult distant metastases.

An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Clinically approved PEGylated nanoparticles are covered by a protein corona that boosts the uptake by cancer cells.

Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been 'opsonization', i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not "stealth" in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.

Biophysics and protein corona analysis of Janus cyclodextrin-DNA nanocomplexes. Efficient cellular transfection on cancer cells.

The self-assembling processes underlining the capabilities of facially differentiated ("Janus") polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications.

A protein corona-enabled blood test for early cancer detection.

Pancreatic cancer is a very aggressive malignancy that is often diagnosed in the advanced stages, with the implication that long-term survivors are extremely rare. Thus, developing new methods for the early detection of pancreatic cancer is an urgent task for current research. To date, nanotechnology offers unprecedented opportunities for cancer therapeutics and diagnosis. The aim of this study is the development of a new pancreatic cancer diagnostic technology based on the exploitation of the nano-bio-interactions between nanoparticles and blood samples. In this study, blood samples from 20 pancreatic cancer patients and 5 patients without malignancy were allowed to interact with designed lipid nanoparticles, leading to the formation of a hard "protein corona" at the nanoparticle surface. After isolation, the protein patterns were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). We found that the protein corona of pancreatic cancer patients was much more enriched than that of healthy individuals. Statistical analysis of SDS-PAGE results allowed us to discriminate between healthy and pancreatic cancer patients with a total discriminate correctness rate of 88%.

Nanoparticles-cell association predicted by protein corona fingerprints.

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.

Exploitation of nanoparticle-protein corona for emerging therapeutic and diagnostic applications.

Exposure of nanoparticles (NPs) to biological fluids (e.g., plasma, interstitial fluid, and cytoplasm) leads to the absorption of proteins on the NP surface, forming a protein corona (PC) that drastically influences the NP physicochemical properties. Herein, we highlight the emerging applications of PC towards its use in therapeutics and diagnostics. In particular, special emphasis is given to the exploitation of PC for targeted delivery of nanomaterials and early cancer detection. By highlighting such recent applications of PC, we hope to demonstrate that this bio-entity has the potential to determine the success of NPs in biomedicine beyond their currently envisioned purposes.

The biomolecular corona of nanoparticles in circulating biological media.

When nanoparticles come into contact with biological media, they are covered by a biomolecular 'corona', which confers a new identity to the particles. In all the studies reported so far nanoparticles are incubated with isolated plasma or serum that are used as a model for protein adsorption. Anyway, bodily fluids are dynamic in nature so the question arises on whether the incubation protocol, i.e. dynamic vs. static incubation, could affect the composition and structure of the biomolecular corona. Here we let multicomponent liposomes interact with fetal bovine serum (FBS) both statically and dynamically, i.e. in contact with circulating FBS (≈40 cm s(-1)). The structure and composition of the liposome-protein corona, as determined by dynamic light scattering, electrophoretic light scattering and liquid chromatography tandem mass spectrometry, were found to be dependent on the incubation protocol. Specifically, following dynamic exposure to FBS, multicomponent liposomes were less enriched in complement proteins and appreciably more enriched in apolipoproteins and acute phase proteins (e.g. alpha-1-antitrypsin and inter-alpha-trypsin inhibitor heavy chain H3) that are involved in relevant interactions between nanoparticles and living systems. Supported by our results, we speculate that efficient predictive modeling of nanoparticle behavior in vivo will require accurate knowledge of nanoparticle-specific protein fingerprints in circulating biological media.

A proteomics-based methodology to investigate the protein corona effect for targeted drug delivery.

Here we introduce a proteomics methodology based on nanoliquid-chromatography tandem mass spectrometry (nanoLC/MS-MS) to investigate the "protein corona effect for targeted drug delivery", an innovative strategy, which exploits the "protein corona" that forms around nanoparticles in a physiological environment to target cells.

The liposome-protein corona in mice and humans and its implications for in vivo delivery.

As soon as nanomaterials, such as nanoparticles (NPs), are injected into a physiological environment a rich coating of biomolecules known as the "protein corona" rapidly covers them. This protein dress is the main factor, which affects the interaction of NPs with living systems. While the relationship between NP features and the biomolecule corona has been extensively investigated, whether and how changes in the physiological environment affect the NP-protein corona remains under-investigated. This is one of the most important steps in translating results in animal models to the clinic. Here we investigated thoroughly the biological identity of lipid NPs (size, charge, aggregation state and composition of the corona) after incubation with human plasma (HP) and mouse plasma (MP) by dynamic light scattering, micro-electrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC/MS-MS). Specifically, we used two different liposomal formulations: the first one was made of polyethyleneglycol (PEG)-coated 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), while the second one was made of 30% of DOTAP, 50% of neutral saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 20% cholesterol. The temporal evolution and complexity of the NP-protein corona was found to be strongly dependent on the biological environment. In MP, liposomes were more negatively charged, less enriched in opsonins and appreciably more enriched in apolipoproteins than their counterparts in HP. Collectively, our results suggest that the biological identities of NPs in mice and humans can be markedly different from each other. Relevance of results to in vivo applications is discussed.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm2/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes.

Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

From small nodule to overt HCC: a multistep process of carcinogenesis as seen during surveillance.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death in the world. Despite many diagnostic and therapeutic tools are now available to improve survival and reduce its recurrence, prognosis is closely conditioned by the time of diagnosis. Surveillance and early diagnosis are crucial for a successful therapy. We report a clinical case from the HCC archive of the Hepatocatt meetings held in Ge-melli Hospital (Catholic University of Rome). The case describes a tumor progression in a multistep process from a small liver nodule to overt HCC and its management by a multidisciplinary team.

Model for end-stage liver disease dynamic stratification of survival benefit.

Only patients with Model for End-stage Liver Disease (MELD) scores ≥18 or ≥17 experience a survival benefit (SB) at 12 and 36 months after liver transplantation (OLT). The SB calculation estimates the difference after stratification for risk categories between the survival rate of transplanted versus waiting list patients. The aim of this study was to perform a short- and long-term (60 months) SB analyses of a Italian OLT program. One-hundred seventy-one patients were stratified into four MELD classes (6-14, 15-18, 19-25, 26-40), and two groups: namely, waiting list (WL) and transplanted groups (TX). The median waiting time for transplanted patients was 4.4 months (range, 0-35). SB was expressed as mortality hazard ratio (MHR) as obtained through a Cox regression analysis using as a covariate the status of each patient in the waiting list (WL = 0, reference group) or the TX group (TX = 1). Values over 1 indicated the MHR in favor of the WL with the values below 1 indicating MHR in favor of Tx. In the MELD class 6 to 14, the MHR was above 1 at 3 and 6 months, indicating an SB in favor of WL; subsequently, the MHR dropped below 1, indicating an SB in favor of TX (P < .05). In the MELD class 15 to 18 the MHR was above 1 at 3 months, but below 1 subsequently (P < .05). For MELD classes 19 to 25 and 26 to 40, the MHR was always below 1 (P < .01). According to the SB approach, patients in the MELD class 6 to 14 could safely wait for at least 36 months; patients in the MELD class 15 to 18 should likely remain no longer than 12 months on the waiting list, and all the remaining patients with MELD > 18 should be transplanted as soon as possible. OLT should not be precluded but only postponed for MELD < 19 patients.

Role of temperature-independent lipoplex-cell membrane interactions in the efficiency boost of multicomponent lipoplexes.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Late development of splenic artery aneurysm after orthotopic liver transplantation: a case report.

Splenic artery aneurysm (SAA) is a rare complication after orthotopic liver transplantation (OLT). Although SAAs are often incidental findings, in some cases they present with signs and symptoms of abdominal mass or intra-abdominal hemorrhage. The diagnosis requires Doppler ultrasound and confirmation with computed tomography, magnetic resonance, or angiography. Endovascular techniques are preferred to surgery for the treatment of most SAAs. A variable interval from 6 days to 11 years has been reported between OLT and the diagnosis of SAA, justifying a lifelong scheduled surveillance of abdominal vessels by ultrasound after OLT. Herein we have reported a case of SAA that developed 16 years after OLT. This pathological condition was totally asymptomatic. Only routine abdominal ultrasound allowed its detection and subsequent successful treatment.

Treatment of chronic hepatitis C virus infection with pegylated interferon and ribavirin in cirrhotic patients awaiting liver transplantation.

Successful treatment of chronic hepatitis C virus (HCV) infection can prevent reinfection after orthotopic liver transplantation (OLT). Pegylated interferon (PEG-IFN) may ameliorate virological response (VR), making the risk-to-benefit ratio of therapy favorable in waiting list patients. From January 2001 to April 2006, we treated 15 HCV cirrhotics with PEG-IFN alpha-2b (1.5 microg/kg/week) and ribavirin (RIBA; >or=10.6 mg/kg/d). Their mean age was 51.5 years. There were 9 men. In 6 cases the genotype was 1b. With Child-Pugh scores >or=9 (range 9-12) and Model for End-Stage Liver Disease (MELD) scores >or=14 (range, 14-22). Adverse events occurred in all subjects: thrombocytopenia (<40,000/microL) in 8; neutropenia (<700/microL) in 10; anemia (Hb <8.5 g/dL) in 1; grade III hepatic encephalopathy in 2; pelvic infection in 1; variceal hemorrhage in 1; and hepatocellular carcinoma (HCC) recurrence in 1. Adverse events caused treatment withdrawal in 6 (40.0%) and RIBA and/or PEG-IFN dose reduction in 10 (66.6%). Early VR (EVR) was obtained in 9 subjects (60.0%), end-of-treatment (EOT) VR in 7 (46.6%), and sustained VR (SVR) in 3 (20.0%). Three subjects--2 nonresponder and 1 breakthrough--were transplanted at 25, 23, and 16 months after the EOT, respectively. Three subjects died at 6, 8, and 15 months after the EOT due to HCC, spontaneous bacterial peritonitis, and liver failure. Nine patients are awaiting OLT. The risk-to-benefit ratio is against PEG-INF and RIBA treatment of severely decompensated cirrhotics infected with genotype 1 awaiting OLT, but therapy is probably beneficial in genotype 2 subjects, due to an expected SVR rate of more than 40%. However, one must carefully consider the high risk for severe adverse events.

Acute decompensation and absence of brain and kidney dysfunction predict long-term efficacy of plasma exchange in hyper-bilirubinemic cirrhotic patients awaiting liver transplantation.

Various artificial liver support systems are currently used in patients with decompensated chronic liver disease or acute liver failure as a bridge to recovery or to orthotopic liver transplantation (OLT). Between June 2004 and September 2006, 9 subjects were treated with plasma exchange (PE) for acute decompensation on chronic liver disease or chronic decompensation in end-stage liver disease. All of them were awaiting OLT or were listed at the moment of decompensation. Grade II to III hepatic encephalopathy (HE) was present in 4 patients, significant renal dysfunction in 3 patients, and ascites in 6 patients. Baseline serum total bilirubin was 35.1+/-11.2 mg/dL (mean value+/-SD). The patients underwent a mean of 12.1 2-hour exchanges over 1 to 8 weeks. The 3 who recovered were alive after a mean follow-up of 22.7+/-10.3 months. There were 3 patients who underwent transplantation and 3 who died due to liver failure during treatment. Only subjects with acute decompensation and without HE or significant renal dysfunction survived without OLT. PE did not significantly modify the grade of HE or the renal function. PE seemed to be a safe, long-term, effective therapeutic option for acute decompensation among subjects with chronic liver disease without brain or renal dysfunction.

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.