Intermolecular interaction studies on native and enzyme-treated acid-soluble collagen.

The aggregative properties of acid-soluble collagen, native or enzyme treated, have been studied by electric birefringence and low shear rate viscosity. A unique type of aggregate has been found, about 700 nm long for native collagen and 530 nm for pepsin treated, regardless of the acetic acid concentration in the range 1--100 mM. The number of aggregates increases with collagen concentrations, as could be expected for electrostatic interactions. On the contrary, pepsin-extracted cartilage collagen forms aggregates of covalent nature, the number of which is independent of concentration. Viscosity measurements show two different interaction mechanisms: a short distance one which can be identified with the electric birefringence-detected aggregation, and a long distance superstructure which disappears when salt is added to the solution.     

Intermolecular interaction studies on native and enzyme-treated acid-soluble collagen

The aggregative properties of acid-soluble collagen, native or enzyme treated, have been studied by electric birefringence and low shear rate viscosity. A unique type of aggregate has been found, about 700 nm long for native collagen and 530 nm for pepsin treated, regardless of the acetic acid concentration in the range 1–100 mM. The number of aggregates increases with collagen concentration, as could be expected for electrostatic interactions. On the contrary, pepsin-extracted cartilage collagen forms aggregates of covalent nature, the number of which is independent of concentration. Viscosity measurements show two different interaction mechanisms: a short distance one which can be identified with the electric birefringence-detected aggregation, and a long distance superstructure which disappears when salt is added to the solution.     

Ovalbumin digestion by human pepsins 1, 3 and 5.

1. Of the three major human pepsins, pepsin 1 has greater proteolytic activity towards ovalbumin than has pepsin 3. Pepsin 5 has low activity towards this substrate. 2. Proteolytic pH-activity curves show only on pH maximum, about pH 1.4 for pepsin 1, pH 1.4--1.5 for pepsin 3 and pH 1.2--1.4 for pepsin 5. The curve for pepsin 3 has a shoulder between pH 2.4 and 3.4. 3. The rate of digestion of ovalbumin by pepsin 1 is approximately three times slower than are those of bovine haemoglobin or human globin. 4. The results suggest that there may be a physiological advantage in having more than one pepsin.     

Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH

Pepsin is an aspartic protease that acts in food digestion in the mammal stomach. An optimal pH of around 2 allows pepsin to operate in its natural acidic environment, while at neutral pH the protein is denatured. Although the pH dependence of pepsin activity has been widely investigated since the 40s, a renewed interest in this protein has been fueled by its homology to the HIV and other aspartic proteases. Recently, an inactive pepsin conformation has been identified that accumulates at mildly acidic pH, whose structure and properties are largely unknown. In this paper, we analyse the conformation of pepsin at different pHs by a combination of spectroscopic techniques, and obtain a detailed characterisation of the intermediate. Our analysis indicates that it is the dominant conformation from pH 4 to 6.5. Interestingly, its near UV circular dichroism spectrum is identical to that of the native conformation that appears at lower pH values. In addition, we show that the intermediate binds the active site inhibitor pepstatin with a strength similar to that of the native conformation. Pepsin thus adopts, in the 6.5-4.0 pH interval, a native-like although catalytically inactive conformation. The possible role of this intermediate during pepsin transportation to the stomach lumen is discussed.     

Does exposure of male Drosophila melanogaster to acute gamma radiation influence egg to adult development time and longevity of F1–F3 offspring?

Two‐ to three‐day‐old male Drosophila melanogaster flies were irradiated with 1, 2, 4, 6, 8, 10, 20, 25, 30, 40 and 50 Gy doses of gamma radiation. The longevity and rate of development were observed for three successive generations to assess the impact of irradiation. The mean lifespan of irradiated flies was significantly increased at 1, 2 and 8 Gy, while it was vice versa for high doses at 30, 40 and 50 Gy. Paternal irradiation had an impact on F₁ generation, with significantly increased mean longevity at 2 (female), 4, 6, 8 and 10 and decreased mean longevity at 40 and 50 Gy (male and female). Significant increase in the longevity was observed in the F₂ generation of the 8 (male and female) and 10 Gy (male) irradiated groups, while decreased longevity was observed in F₂ female progeny at 40 Gy. In the case of F₃ progeny of irradiated flies, longevity did not show significant difference with the control. Paternal exposure to radiation had a significant impact on the mean egg to adult developmental time of the F₁ generation; it was shortened at 2 Gy and extended at 25, 30, 40 and 50 Gy compared to the control. Mean development time at 30, 40 and 50 Gy was significantly increased in the F₂ generation, while there were no significant changes in the F₃ generation. The present study concludes that the effect of acute gamma irradiation on longevity and “egg to adult” development time of D. melanogaster may persist to following generations.     

Reversible association of proteins into sub-visible amorphous aggregates using short solubility controlling peptide tags

Careful analysis of sub-visible amorphous aggregates, where proteins associate non-covalently in either native or denatured states without forming a specific quaternary structure, may shed insight into the mechanisms of protein aggregation and solubility. Here we report a biophysical and biochemical analysis of our model protein, a bovine pancreatic trypsin inhibitor variant (BPTI-19A), whose oligomerization were controlled by attaching solubility controlling peptide tags (SCP tags) to its C terminus, which are short peptides composed of a single type of amino acid that modulate protein solubility. The dynamic light scattering and static light scattering at 25 °C indicated that 11 out of 15 SCP tags merely affected the hydrodynamic radius and light scattering intensity of our reference variants BPTI-19A and BPTI-C2G. On the other hand, hydrophobic SCP tags composed of 5 Ile (C5I) or 5 Leu (C5L) were associated into sub-visible aggregates. Circular dichroism indicated that all tagged BPTI variants had the same secondary structure contents as the reference BPTI-19A at 25 °C, suggesting that BPTI-C5I and C5L kept their native structure upon association. Furthermore, the thermal denaturation of all of the BPTI variants was fully reversible and typical of natively folded small globular proteins, as monitored by CD at 222 nm. However, the thermal stability of BPTI-19A tagged with hydrophobic residues decreased with increasing protein concentration and tag's hydrophobicity, and BPTI-C5I and C5L were partially denatured at 37 °C. Biochemical stability assessed by limited proteolysis with pepsin correlated with the extent of the variants' aggregation, and the large sub-visible aggregates formed by BPTI-C5I and C5L significantly increased their resistance to pepsin proteolysis. Altogether, these observations indicated that hydrophobic SCP tags led to the reversible association of native-like proteins into sub-visible soluble amorphous aggregates resistant to pepsin digestion.     

Intermolecular association and molecular weight of aggregates of a monodisperse homohexapeptide,Nps-[Glu(OBzl)]6-NHEt,in solution

Light scattering from the solutions of Nps-[Glu(OBzl)]₆-NHEt in dioxane or ethylene dichloride has been measured at different concentrations, and a critical concentration of intermolecular association is found to exist, which is equal to the critical concentration of β-form formation. The Debye plot of light scattering leads to the molecular weight of aggregates at the critical concentration, which corresponds to an aggregation number 15 in dioxane and 53 in ethylene dichloride. In the latter solvent the aggregates further associate into a larger aggregate consisting of 330 molecules when the concentration is increased beyond the critical concentration. The content of β-form, which is a measure of number of hydrogen bonds, is derived from the ir data previously obtained. The results on the modes of intermolecular association and hydrogen bonding lead to possible structures of aggregates formed by both hydrogen bonds and other nonbonding side-chain interactions.     

A slow fluctuation in the molecular conformation of a soya bean trypsin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a trypsin inhibitor (Kunitz) from soya bean have been followed by infrared absorption measurements in aqueous solutions at various temperatures and pH values. It was found that, in every case, 49% of the total peptide hydrogen atoms exchange relatively slowly. This amount corresponds to 83 peptide groups per molecule, and this is considered to be equal to the number of peptide NH groups involved in hydrogen bonds with the carbonyls of other peptide groups in the protein molecule in its native form. Each rate constant (k) determined at pH 2.75 for this category of the NH groups is in good agreement with the value expected from an idea that the breaking of the peptide-peptide hydrogen bonds takes place very slowly, and that this is the rate-determining process in the hydrogen-deuterium exchange reaction. Thus, by ultraviolet absorption measurements at 297 nm, the equilibrium constant of the native and denatured forms has been determined in the temperature range from 42 to 53.5 °C, as well as the reaction rate of reaching equilibrium from an off-equilibrium state. From these data the rate constant (k₁) of the denaturation reaction is determined, and the k₁ value is found to be practically equal to the hydrogen exchange rate constant (k). The Arrhenius plot of this rate constant (k) gives a straight line in the 25 to 55 °C region, and this gives a value of 48.6 kcal/mol for the activation energy of the denaturation reaction. The rate of this reaction is found to be very low at 25 °C; its half-life is about eleven days. Infrared absorption spectra observed in the amide I region suggest that the very slow denaturation of this protein is accompanied by a conformation change from an α-helix to a β-form. The number of the peptide groups involved in this α → β change is estimated to be 9 ± 3.     

Relationship between functional properties and structure of ovalbumin

The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHCl), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and 5 different cationic detergents with various side chains, HCl, and CH₃COOH were observed. Progressive unfolding in ovalbumin was measured as a function of fluorescent light intensity, peak response and shift in the maximum of emission. Kinetic measurements demonstrated that the rate of denaturation usually followed a double exponential decay pattern, but at small concentrations of urea and acids first-order reaction was indicated. The reversibility of the unfolding-folding transitions was confirmed from tryptophan fluorescence and circular dichroism (CD) measurements. Differences in secondary structure were observed and changes of-helical content were calculated. Polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulphate (SDS-PAGE) showed differences in the structure of native and denatured ovalbumin. Native protein samples in PAGE demonstrated smaller number and larger mobilities of subunits than denatured ones with different reductants, such as SDS and 2-mercaptoethanol (2 ME). Scanning of SDS protein patterns showed the appearance of aggregated forms in region of 45 kD.     

Solution properties of porcine submaxillary mucin

Porcine submaxillary mucin (PSM) is a glycoprotein composed of a protein core and frequent, short oligosaccharide side chains. We report static and dynamic light scattering experiments and intrinsic viscosities for PSM in aqueous solvent systems. In 0.1M NaCl solution, the data suggest PSM exists as large, internally branched, highly hydrated, polydisperse aggregates that slowly dissociate to give a stable species of weight-average molecular weight (M_w) 7.4 × 10⁶. In 6M GdnHCl solution, the noncovalent bonds between PSM molecules are broken, giving a highly elongated molecule of M_w = 2.0 × 10⁶. The irreversible nature of this dissociation suggests that the forces that stabilize the native aggregates of PSM in 0.1M NaCl are specific in nature. On reduction of PSM with mercaptoethanol, the polydispersity decreases and M_w also decreases to 9 × 10⁵. A discrete change is observed in the solution properties of PSM in 0.1M NaCl at a concentration of 2mg/mL, manifested by a sudden decrease in the translational diffusion coefficient, an increase in viscosity number, and a decrease in slope of the osmotic compressibility. We tentatively propose that a weak and reversible secondary association process occurs at this concentration, although a purely hydrodynamic interaction cannot be ruled out.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

Blood Thixotropy in Patients with Sickle Cell Anaemia: Role of Haematocrit and Red Blood Cell Rheological Properties

We compared the blood thixotropic/shear-thinning properties and the red blood cells’ (RBC) rheological properties between a group of patients with sickle cell anaemia (SS) and healthy individuals (AA). Blood thixotropy was determined by measuring blood viscosity with a capillary viscometer using a “loop” protocol: the shear rate started at 1 s⁻¹ and increased progressively to 922 s⁻¹ and then re-decreased to the initial shear rate. Measurements were performed at native haematocrit for the two groups and at 25% and 40% haematocrit for the AA and SS individuals, respectively. RBC deformability was determined by ektacytometry and RBC aggregation properties by laser backscatter versus time. AA at native haematocrit had higher blood thixotropic index than SS at native haematocrit and AA at 25% haematocrit. At 40% haematocrit, SS had higher blood thixotropic index than AA. While RBC deformability and aggregation were lower in SS than in AA, the strength of RBC aggregates was higher in the former population. Our results showed that 1) anaemia is the main modulator of blood thixtropy and 2) the low RBC deformability and high RBC aggregates strength cause higher blood thixotropy in SS patients than in AA individuals at 40% haematocrit, which could impact blood flow in certain vascular compartments.     

The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO₂)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.     

The C-terminal extrahelical peptide of type I collagen and its role in fibrillogenesis in vitro

The formation in vitro of fibrils from type I acid-soluble calf skin collagen has been studied before and after removal of the extrahelical peptides with carboxypeptidase and with pepsin. Turbidimetric studies show that the mechanism of fibril growth in undigested collagen is similar to that in pepsin-digested collagen; following carboxypeptidase digestion, however, a different growth mechanism was apparent. The two mechanisms have been further characterized by electron microscopy. In the course of formation of fibrils from undigested collagen, “early fibrils” (short D-periodic fibrils that have both ends visible) occurred in the lag phase under the precipitating conditions employed here. After pepsin or carboxypeptidase digestion of the collagen no “early fibrils” were seen. In carboxypeptidase-digested collagen, lateral assembly was inhibited; after pepsin digestion, linear assembly was inhibited. Complete removal of the extrahelical peptides prevented fibril formation under the conditions used here. Electron-optical examination of segment-long-spacing (SLS) dimers established a more complete removal of the C-terminal peptide after carboxypeptidase digestion than after pepsin digestion. Analyses of staining patterns of SLS dimers and fibrils from undigested and digested samples showed that the C-terminal peptide in SLS crystallites and fibrils formed from undigested collagen is in a condensed conformation. A proposed conformation, in which condensation occurs predominantly in a hydrophobic region at the proximal end of the C-terminal peptide, is discussed in terms of a dual role for the C-terminal peptide in fibrillogenesis. One role, shared with the N-terminal peptide, is to participate in interactions between the 4D-staggered molecules leading to the formation of linear aggregates; the other is to participate in interactions between these linear aggregates giving rise to D-periodic aggregates and lateral (as well as linear) growth.     

Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations

The conformation of heat-denatured ovalbumin aggregates has been examined at several concentrations and pH values, using measurements of optical rotation dispersion (ORD), circular dichroism (CD) and viscosity. The protein was subjected to heating at relatively low temperatures, ranging from 48.5 to 76 degrees; the particular temperature chosen depended on pH. The heat treatment was sufficient to remove the ability of the molecules to absorb heat on re-heating. The denatured molecules were shown to be rather compact, i.e. not much larger than the native molecule, and to retain a significant amount of secondary structure; this was also the case for molecules present in small aggregates. It is suggested that this type of ovalbumin monomer builds three-dimensional networks in denatured solutions at higher concentrations, and that gelation should be looked upon as arising from surface contacts between hydrated globules. The present results also imply that such globules have gelation properties which depend on whether pH is acidic or basic compared to the isoelectric point of the protein.     

REPOPULATION OF THE BONE-MARROW STEM-CELL COMPARTMENT AFTER IRRADIATION*

Bone-marrow repopulation has been followed as a function of time after a single whole-body dose of 150 rads X-rays to (C3H × C57B1)F₁ hybrid mice. The number of nucleated cells per bone shaft has been estimated by direct counting and the number of progenitor cells by the exogenous spleen-colony assay method. Vinblastine was also administered under various schedules of time and dose in an endeavour to distinguish between cycling and dormant cells in the progenitor pool. The depopulation of total marrow cells induced by Vinblastine proceeds at a comparable rate in irradiated and in normal mice. On the other hand, the depopulation of the colony-formers is faster in irradiated than in normal animals. The data are interpreted to show that the fraction of cycling progenitor cells is larger in a haemopoietic tissue undergoing numerical expansion.     

Kerr effect study of the aqueous solutions of three globular proteins

A Kerr effect study is reported in which measurements have been made on the magnitudes of both the steady maxima and the decays of the birefringence of solutions of ovalbumin, bovine γ-globulin, and β-lactoglobulin. For each protein, results are presented on solutions covering the concentration range of 0.3–1.7 g./100 ml. in order to obtain by extrapolation, values of the specific Kerr constant K_sp, and the birefringence relaxation time τ_{25, w} at zero concentration. The relaxation times thus obtained for ovalbumin (18.3 nsec.) and γ-globulin (157 nsec.) have been shown to be compatible with molecular models and dimensions presented in the literature. All experiments showed the need for careful extrapolation to zero concentration if reliable parameters are to be obtained: for example a 1% solution of ovalbumin or l.5% solution of γ-globulin, would give values for τ which are 50% too high when compared with the true value at infinite dilution. The gradual fall in τ for γ-globulin as the pH was lowered from 6.7 to 3.0 was also studied for three solvents. Fisher's generalized model for the arrangement of the polar residues around the outside of a globular protein has been developed to account for ellipsoidal particles and has been used to demonstrate the suitability and usefulness of this treatment in predicting the conformation and dimensions of these proteins. Rather unusual birefringence traces for β-lactoglobulin were obtained, which may indicate the dissociation of aggregates, or of the parent molecule into its subunits, under the influence of strong electric fields.     

Partial denaturation of macromolecules by chemical agents

The denaturation of a macromolecule such as a polypeptide is considered in the case where the total number of noncovalent internal bonds broken in the binding process of the chemical agent is only a fraction, α_s, of the total number of noncovalent internal bonds involved in its helical native conformation. Starting from a two-parameter (s, σ) model of the helix–random coil transition of polypeptides, the transition temperature is derived as a function of the fraction α_s, and of the concentration and the binding constant of the chemical agent. The lower limit of the transition temperature and the corresponding slope of the transition curve are shown to depend on α_s. As an illustration, existing data on the partial acid denaturation of the enzymes ribonuclease A and muromidase are analyzed. The resulting average enthalpies of a noncovalent bond in the native ribonuclease A and muromidase are found to be 1.81 kcal/mole and 1.54 kcal/mole, respectively; the corresponding entropies are 5.4 and 4.4 cal/deg./mole, respectively. As a further example, existing data on a partially methylated enzyme ribonuclease A are also considered.     

Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections

Estrogen has been shown to affect ventromedial hypothalamic (VMH) nerve cell nucleoli in ovariectomized rats, by causing an increase in the number of electron-dense aggregates associated with nucleoli. In order to characterize these nucleolus-associated structures and other nuclear components, we examined the ultrastructure of ventromedial hypothalamic nucleoli and nuclei revealed by enzyme digestions (pepsin, RNase and DNase) in resinless thin sections. Digestion by pepsin did not cause obvious alterations in the morphology of the nucleolus or its related structures. Pepsin treatment followed by RNase, however, reduced the density of the nucleolus, while that of the nucleolus-associated structure and other related structures remained unchanged. Conversely pepsin treatment followed by DNase, reduced the density of nucleolus-associated and other chromatin structures, but had no effect on the density of the nucleolus. Pepsin treatment followed by RNase and then DNase treatment, reduced the density of the nucleolus and nucleolus-associated structures. A residual nucleolus and nucleolus-associated structure remained after this treatment. Stereo viewing of resinless sections shows that the nucleolus, its associated structures, and other related structures, are associated with fine filaments that may comprise the nuclear matrix. The nucleolus-associated structure containing DNA may direct RNA synthesis at an increased rate in estrogen-treated hypothalamic cells.