Proteins

O presente trabalho teve por objetivos avaliar a composição de rizomas do lírio-do-bejo (Hedychium coronarium) e isolar e caracterizar seu amido, por meio de análises físicas e físico-químicas. A composição química dos grânulos de amido isolados dos rizomas também foi determinada, bem como o formato, tamanho, distribuição de tamanho de partícula por difração a laser, viscosidade de pasta em analisador rápido de viscosidade, poder de inchamento, índice de solubilidade em água, propriedade térmica por calorimetria diferencial de varredura e cristalinidade por difração de raios X. Observou-se que os rizomas apresentaram elevado teor de água e baixos teores de matéria graxa, carboidratos, cinzas, proteínas e fibras. Os rizomas de lírio-do-brejo apresentam-se inadequados para produção de amido, devido ao baixo teor de matéria seca (17,3%), com 21,97% de amido, bem como fibras longas e látex, que dificultam a extração do amido. O amido extraído apresentou pureza de 99,2%, com 37,2% de ami...

A full quantitative understanding of the protein folding problem is now becoming possible with the help of the energy landscape theory and the protein folding funnel concept. Good folding sequences have a landscape that resembles a rough funnel where the energy bias towards the native state is larger than its ruggedness. Such a landscape leads not only to fast folding and stable native conformations but, more importantly, to sequences that are robust to variations in the protein environment and to sequence mutations. In this paper, an off-lattice model of sequences that fold into a ␤-barrel native structure is used to describe a framework that can quantitatively distinguish good and bad folders. The two sequences analyzed have the same native structure, but one of them is minimally frustrated whereas the other one exhibits a high degree of frustration.

Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for 1 H and showed that the underlying dynamics driving the protein 1 H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of 1 H 2 O in the total proton-spin-relaxation process. The fact that the proteinproton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with 2 H 2 O rather than 1 H 2 O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by 1 H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements.

Structural information on soil organic matter (SOM) at the molecular level can be obtained on diverse structural units that are amenable to degradation techniques. Chemolytic techniques in combination with colorimetric analyses or GC MS are used to determine amino acids (proteins), sugars (polysaccharides), lipids, or aromatic oxidation products from lignin or charred organic matter. Microbial markers (amino sugars, muramic acid) are analyzed after hydrolysis and gas chromatographic separation. Macromolecular structures can also be subjected to thermochemolytic degradation or pyrolysis and subsequent analysis of the fragments by GC MS. Alternative techniques for the examination of organic matter in heterogeneous macromolecular mixtures are non-destructive spectroscopic methods, such as nuclear magnetic resonance (NMR) spectroscopy. Although this technique can give good results concerning the gross chemical composition, speci®c compounds are hardly identi®ed. The combination of spectroscopic techniques with thermolytic and chemolytic methods will add substantially to the understanding of the nature of refractory soil organic matter. Physical fractionation prior to analysis provides a means to dierentiate between distinct SOM pools that can be further characterized by the methods described above. Studies on SOM structural characteristics have focused mainly on the A horizons of soils under agriculture and litter biodegradation in forest soils and need to be extended to a wider variety of soil types and the subsoil. #

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 express...

A biosensor is an analytical device that consists of an immobilized biocomponent in conjunction with a transducer, and represents a synergistic combination of biotechnology and microelectronics. This review summarizes the use of biosensors for detecting and quantifying heavy metal ions. Heavy metal contamination is of serious concern to human health since these substances are non-biodegradable and retained by the ecological system. Conventional analytical techniques for heavy metals (such as cold vapour atomic absorption spectrometry, and inductively coupled plasma mass spectrometry) are precise but suffer from the disadvantages of high cost, the need for trained personnel and the fact that they are mostly laboratory bound. Biosensors have the advantages of specificity, low cost, ease of use, portability and the ability to furnish continuous real time signals. The analysis of heavy metal ions can be carried out with biosensors by using both protein (enzyme, metal-binding protein and antibody)-based and whole-cell (natural and genetically engineered microorganism)-based approaches.

An electrophoretic and microbiological study [more probable number (NMP), total (CT) and fecal coliforms (CF); aerobic-mesophils bacteria (BAM), molds andyeasts] in worm's flour of Eisenia fétida was carried out. In the electrophoretic proflles using gels of ...

Mycotherapy is defined as the study of the use of extracts and compounds obtained from mushrooms as medicines or health-promoting agents. The present review updates the recent findings on anticancer/antitumor agents derived from mushroom extracts and their metabolites. The increasing number of studies in the past few years revealed mushroom extracts as potent antitumor agents. Also, numerous studies were conducted on bioactive compounds isolated from mushrooms reporting the heteropolysaccharides, -glucans, -glucans, proteins, complexes of polysaccharides with proteins, fatty acids, nucleoside antagonists, terpenoids, sesquiterpenes, lanostanoids, sterols and phenolic acids as promising antitumor agents. Also, molecular mechanisms of cytotoxicity against different cancer cell lines are discussed in this review. Findings with Antrodia camphorata and Ganoderma lucidium extracts and isolated compounds are presented, as being the most deeply studied previously.

Ridden .Markov Models (HMMs) are applied t.0 the problems of statistical modeling, database searching and multiple sequence alignment of protein families and protein domains. These methods are demonstrated on the globin family, the protein kinase catalytic domain, and the EF-hand calcium binding motif. In each case the parameters of an HMM are estimated from a training set of unaligned sequences. After the HMM is built, it is used to obtain a multiple alignment of all the training sequences. It is also used to search the . SWISS-PROT 22 database for other sequences. that are members of the given protein family, or contain the given domain. The Hi" produces multiple alignments of good quality that agree closely with the alignments produced by programs that incorporate threedimensional structural information. When employed in discrimination tests (by examining how closely the sequences in a database fit the globin, kinase and EF-hand HMMs), the '\ HMM is able to distinguish members of these families from non-members with a high degree ' of accuracy. Both the HMM and PROFILESEARCH ( a technique used to search for relationships between a protein sequence and multiply aligned sequences) perform better in these tests than PROSITE (a dictionary of sites and patterns in proteins). The HMM appecvs to have a slight advantage over PROFILESEARCH in terms of lower rates of false negatives and false positives, even though the HMM is trained using only unaligned sequences, whereas PROFILESEARCH requires aligned training sequences. Our results suggest the presence of an EF-hand calcium binding motif in a highly conserved and evolutionary preserved putative intracellular region of 155 residues in the a-l subunit of L-type calcium channels which play an important role in excitation-contraction coupling. This region has been suggested to contain the functional domains that are typical or essential for all L-type calcium channels regardless of whether they couple to ryanodine receptors, conduct ions or both. \ 8 Abbreviations HMM, hidden Marko,. ,,,dels; length from 130 to 170 residues (with few excep-' m, Expectetion-Maximization; ML. maximum tions) and two domains, the protein kinase catalytic like€ihood; MAP, maximum a posten'wi; hTL-score.

We investigate the potential of fluorescence correlation spectroscopy (FCS) in screening for crystallisation conditions. Solutions that nucleate protein crystals must have different interactions than solutions that do not give rise to crystals. Due to these different interactions the average mean squared displacement of the individual proteins changes. By monitoring protein self-diffusion, we can distinguish crystallising from non-crystallising solutions. The method introduced can be applied at extremely low concentrations in femtoliter volumes as an early diagnostic for molecular association. Based on our preliminary findings FCS has the potential to become a routine screening method for crystallography.

The present study characterizes the dynamic interfacial properties of calf lung surfactant (CLS) and samples reconstituted in a stepwise fashion from phospholipid (PL), hydrophobic apoprotein (HA), surfactant apoprotein A (SP-A), and neutral lipid fractions. Dipalmitoylphosphatidylcholine (DPPC), the major PL component of surfactant, was examined for comparison. Surface tension was measured over a range of oscillation frequencies (1-100 cycles/min) and bulk phase concentrations (0.01-1 mg/ml) by using a pulsating bubble surfactometer. Distinct differences in behavior were seen between samples. These differences were interpreted by using a previously validated model of surfactant adsorption kinetics that describes function in terms of 1) adsorption rate coefficient (k1), 2) desorption rate coefficient (k2), 3) minimum equilibrium surface tension (gamma*), 4) minimum surface tension at film collapse (gammamin), and 5) change in surface tension with interfacial area for gamma < gamm...

High pressure homogenization (HPH) was tested for inducing autolysis in a commercial strain of Saccharomyces bayanus for winemaking. The effects on cell viability, the release of soluble proteins, glucidic colloids and amino acids in wine-like medium and the volatile composition of the autolysates were investigated after processing, in comparison with thermolysis. HPH seemed a promising technique for inducing autolysis of wine yeasts. One pass at 150MPa was the best operating conditions. Soluble colloids, proteins and free amino acids were similar after HPH and thermolysis, but the former gave a more interesting volatile composition after processing, with higher concentrations of ethyl esters (fruity odors) and lower fatty acids (potential off-flavors). This might allow different winemaking applications for HPH, such as the production of yeast derivatives for wine ageing. In the conditions tested, HPH did not allow the complete inactivation of yeast cells; the treatment shall be optimized before winemaking use.

Ascites can be defined as the abnormal accumulation of fluid inside the peritoneal cavity. Earlier ascites was classified as transudative and exudative based on the total protein concentration of the ascitic fluid. The traditional concept of high pro-tein ascites(> 2.5 g/dl) as exudate ...

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal-Galactosidase (-Gal) content is described. Cells were transported single file toward a Y-shaped mixing junction at which lytic agents were introduced by suction. Flow velocities of 100 and 40 m/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaturing (0.1% Triton X-100) conditions, respectively. Complete and reproducible lysis of individual cells on-chip occurred within 30 s using Triton X-100 and 2 s when using SDS. Optimal concentrations of lysis and enzyme substrate reagents were determined using microtitre plate and chip-based procedures. Fluorescence peaks, due to the enzymatic product fluorescein mono-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of-Gal with 200 M of the fluorogenic substrate fluorescein-diD -galactopyranoside (FDG). FMG fluorescence was observed from cells preincubated with FDG off-chip then subsequently lysed on-chip with SDS. Unincubated cells were mixed on-chip with both FDG and Triton X-100, each individual cell generating FMG fluorescence downstream of the mixing point detected within 2 min of mixing. In contrast, viable cells incubated with FDG required 1 h or more in order to generate significant signal in a flow cytometer.

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Parathyroid hormone-related protein (PTHrP) is the major mediator of the humoral hypercalcemia of malignancy and of malignant osteolysis associated with skeletal metastases of common epithelial cancers. PTHrP secretion is regulated by the extracellular calcium concentration ([Ca(2+)](o)) in several types of normal and malignant cells. Because the [Ca(2+)](o)-sensing receptor (CaR) is a key mediator of [Ca(2+)](o)-regulated hormone secretion [e.g., of parathyroid hormone (PTH) by parathyroid chief cells], we investigated the expression of the CaR and PTHrP in normal and neoplastic glial cells and studied the effects of [Ca(2+)](o) on PTHrP secretion. Our results show that primary embryonic human astrocytes (HPA) express CaR mRNA and protein as detected by RT-PCR and Western analysis, respectively. Furthermore, astrocytomas and meningiomas also express the CaR at similar levels as assessed by RT-PCR and Northern and Western blot analyses. HPA and astrocytomas express transcripts encod...

creted 23.9% after the oral dose. Thus, based on urinary excretion of total radioactivity in dogs, it appears that only 5040% of furosemide is absorbed from an oral solution. The plasma and tissue levels and the pharmacokinetic parameters of furosemide in dogs and monkeys following 5-mg/kg oral and intravenous doses will be described in a subsequent paper. These data in dogs confirm the lack of complete bioavailability of furmmide from an oral solution dosage form. Less than 1% of radioactivity in the plasma of dog and monkey is attributable to metabolites.

An efficient molecular simulation methodology has been developed to determine the positioning of water molecules in the binding site of a protein or protein-ligand complex. Occupancies and absolute binding free energies of water molecules are computed using a statistical thermodynamics approach. The methodology, referred to as JAWS, features "θ-water" molecules that can appear and disappear on a binding-site grid. Key approximations render the technique far more efficient than conventional free energy simulations. Testing of JAWS on five diverse examples (neuraminidase, scytalone dehydratase, major urinary protein 1, β-lactoglobulin, and COX-2) demonstrates its accuracy in locating hydration sites in comparison to results from high-resolution crystal structures. Possible applications include aid in refinement of protein crystal structures, drug lead optimization, setup of docking calculations and simulations of protein-ligand complexes.

Successful use of liposomes as immunological adjuvants in vaccines requires simple, easy to scale up technology capable of high-yield antigen entrapment. Recent work from this laboratory has led to the development of techniques that can generate liposomes of various sizes containing soluble antigens such as proteins or particulate antigens such as whole, live, or attenuated bacteria or viruses. Entrapment of proteins is carried out by the dehydration-rehydration procedure, which entails freeze-drying of a mixture of "empty" small unilamellar vesicles and free antigens. Upon rehydration, the large multilamellar vesicles that are formed incorporate up to 80% of the antigen used. When such liposomes are microfluidized in the presence of nonentrapped material, their size is reduced to about 100 nm in diameter, with much of the originally entrapped antigen still associated with the vesicles. A similar technique applied to the entrapment of particulate antigens (e.g., Bacillus s...

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 WM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide. ß

Although the precise mechanisms explaining loss of, and failure to regain, function after spinal cord injury are unknown, there is increasing interest in the role of "secondary cell death." One prevalent theme in cell loss in other regions of the CNS involves apoptosis executed by the intracellular caspase proteases. A recent study demonstrated that spinal cord injury rapidly increased the activation of caspase-3. Our previous studies demonstrated peak apoptosis in three of four cellular compartments 3 days after controlled contusion in the rat. We have extended these analyses to include enzyme and substrate studies of caspase subfamilies both in rostral and in caudal adjacent segments compared to the lesion site. Although presumed activation of programmed proenzyme is considered the mechanism for enhanced caspases, our novel analyses were designed to detect upregulation of gene expression. We surveyed traumatically injured spinal cord for caspase family messages with a modified differential mRNA display approach and found that the caspase-3 (CASP3) message was present and upregulated severalfold after injury. Our results clearly demonstrate that cell death in the spinal cord occurs after posttranslational activation of caspases that follow, at least for caspase-3, initial upregulation of CASP3 mRNA levels.

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

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Tumors evade host immunity at both the induction and effector phases. Most studies have focused on tumor evasion at the induction phase, and, due in part to poor antitumor CTL responses to most tumors, the mech- anism for evasion of CTL effector function is less clear. Here we have taken advantage of the strong CTL responses to a costimulator B7-1- transfected tumor to study the mechanism for tumor evasion of preexist- ing host immunity. We have investigated six independent recurrent tu- mors isolated from mice that were challenged with and had rejected B7-1-transfected J558 (J558-B7) tumors. Because the mice had developed strong antitumor CTL responses, these recurrent tumors must have evaded preexisting antitumor CTLs. Indeed, whereas the parental J558-B7 cell line is efficiently lysed by the ex vivo tumor-infiltrating lymphocytes, all of the recurrent tumors are resistant to such lysis. Interestingly, the recurrent tumors can be divided into two groups. The group 1 tumors have va...

Helix kinks are a common feature of a-helical membrane proteins, but are thought to be rare in soluble proteins. In this study we find that kinks are a feature of long a-helices in both soluble and membrane proteins, rather than just transmembrane ahelices. The apparent rarity of kinks in soluble proteins is due to the relative infrequency of long helices (20 residues) in these proteins. We compare length-matched sets of soluble and membrane helices, and find that the frequency of kinks, the role of Proline, the patterns of other amino acid around kinks (allowing for the expected differences in amino acid distributions between the two types of protein), and the effects of hydrogen bonds are the same for the two types of helices. In both types of protein, helices that contain Proline in the second and subsequent turns are very frequently kinked. However, there are a sizeable proportion of kinked helices that do not contain a Proline in either their sequence or sequence homolog. Moreover, we observe that in soluble proteins, kinked helices have a structural preference in that they typically point into the solvent.

The aim of this work was to identify a new protein that discriminated CD8 from CD4 and CD19 lymphocytes. Proteins were separated by high-resolution two-dimensional electrophoresis. After silver staining, the gel images were captured with a laser densitometer, and studied with a dedicated software. This study confirmed the presence of two spots that appeared to be preferentially associated with CD8 lymphocytes, and mass spectrometry analyzes (liquid chromatography-tandem mass spectrometry, LC-MS/MS) identified six peptides for one spot and four for the other. The peptide sequences corresponded to an unknown protein that we named swiprosin 1 (Swiss-Prot Q96C19). Molecular analysis (reverse transcriptase-polymerase chain reaction, RT-PCR) and Northern blots confirmed that the gene expression was increased in purified populations of CD8 lymphocytes, when compared to CD19 and CD4 lymphocytes. Database mining revealed that swiprosin 1 contains two potential EF-hand domains, and therefore may have a role in calcium signaling. Its predominant presence in CD8 lymphocytes suggests that it may be involved in functions that are important for cytotoxic lymphocytes.

We used immunohistochemistry and electron microscopy to evaluate the differentiation of cells comprising atypical adenomatous hyperplasia (AAH; n = 26), early bronchioloalveolar lung carcinoma (BAC; n = 11), and overt BAC (n = 16), which are assumed to constitute a continuous spectrum of developmental steps of BAC. Surfactant apoprotein (SAP), a marker for type 2 alveolar cells, was expressed in cells from all the lesions of AAH, early BAC, and overt BAC. However, the proportion of SAP-positive cells decreased and their distribution became more heterogeneous with advancing lesion grade. Urine protein 1, which is identical to the Clara cell-specific 10 kDa protein, was expressed in 70% of overt BAC, whereas only 20% of early BAC showed weak reactivity and none of AAH lesions showed any reactivity at all. Ultrastructurally, type 2 alveolar cell differentiation was predominant among cells from AAH and early BAC. Our results suggest that precursor cells of BAC differentiate predominantly towards type 2 alveolar cells. Cells comprising overt BAC retain this differentiation phenotype, but to a reduced extent. In contrast, concomitantly with progression, cells with Clara cell differentiation emerge and their proportion increases. Such phenotypic changes may reflect metaplasia occurring in tumour cells during the development of BAC.

Neuronal growth cones navigate over long distances along specific pathways to find their correct targets. The mechanisms and molecules that direct this pathfinding are the topics of this review. Growth cones appear to be guided by at least four different mechanisms: contact ...

We report here a robust automated active site detection, docking, and scoring (AADS) protocol for proteins with known structures. The active site finder identifies all cavities in a protein and scores them based on the physicochemical properties of functional groups lining the cavities in the protein. The accuracy realized on 620 proteins with sizes ranging from 100 to 600 amino acids with known drug active sites is 100% when the top ten cavity points are considered. These top ten cavity points identified are then submitted for an automated docking of an input ligand/candidate molecule. The docking protocol uses an all atom energy based Monte Carlo method. Eight low energy docked structures corresponding to different locations and orientations of the candidate molecule are stored at each cavity point giving 80 docked structures overall which are then ranked using an effective free energy function and top five structures are selected. The predicted structure and energetics of the complexes agree quite well with experiment when tested on a data set of 170 proteinÀligand complexes with known structures and binding affinities. The AADS methodology is implemented on an 80 processor cluster and presented as a freely accessible, easy to use tool at http://www.scfbio-iitd.res.in/ dock/ActiveSite_new.jsp.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermo-dynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 105 M–1) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 108 M–1); (ii) LDTI be-haves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine tryp-sin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 108 M–1). Amino acid sequence alignment of BLCT, human II-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-...

The UV photolysis of the aromatic amino acid, tryptophan (Trp), in the Ca2+-binding protein, cod parvalbumin, type 111, was studied using electron paramagnetic resonance (EPR) spectroscopy in the temperature range 4-80 K. For the Ca2+-bound protein, irradiation with UV light (250-400 nm) resulted in the generation of atomic hydrogen with a hyperfine splitting of 50.9 mT, whereas in the Ca2+-free form, where the Trp is exposed to solvent, the trapped atomic hydrogen was not in evidence. In the same spectra, the radical signal in the g = 2.00 region could be detected. The line shape of the Ca2+bound form is similar to the EPR line shape obtained for Trp in micellar systems. In contrast, the EPR line shape for the Ca2+-free form is essentially featureless up to 80 K. The EPR spectra of the photoproducts of Trp and the nature of the photoreactions are therefore sensitive to the environment of Trp within the protein.

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4000Å) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pHfractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.

Looking at proteins is an active process of interpretation and selection, emphasizing some features and deleting others. Multiple representations are needed, for such purposes as showing motions or conveying both the chain connectivity and the three-dimensional shape simultaneously. In studying and comparing protein structures, ideas are suggested about the determinants of tertiary structure and of folding (e.g., that Greek key #3 barrels may fold up two strands at a time). The design and synthesis of new proteins "from scratch" provides a route toward the experimental testing of such ideas. It has also been a fruitful new perspective from which to look at structures, requiring such things as statistics on very narrowly defined structural categories and explicit attention to "negative design" criteria that actively block unwanted alternatives (e.g., reverse topology of a helix bundle, or edge-to-edge aggregation ofs heets). Recently, the field of protein design has produced a rather unexpected general result: apparently we do indeed know enough to successfully design proteins that fold into approximately correct structures, but not enough to design unique, native-like structures. The degree of order varies considerably, but even the best designed material shows multiple conformations by NMR, more similar to a "molten globule" folding intermediate than to a well ordered native tertiary structure. In response to this conclusion, we are now working on systems that test useful questions with approximate structures (such as determining which factors most influence the choice of helix-bundle topology) and also analyzing how natural proteins achieve unique core conformations (e.g., for side chains on the interior side of a (3 sheet, illustrated in the kinemages).

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 C, 20 C, 30 C and 40 C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 C, 20 C and 30 C. Interleukin-6 concentrations significantly declined after 11 days at 40 C. The concentrations of sIL-6R and IL-10 did not change at 4 C but significantly decreased at 20 C (after 21 and 14 days respectively), 30 C and 40 C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at 20 C and 70 C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 C or 20 C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at 20 C for several years, but for IL-10 determinations, storage at 70 C is recommended.