Antioxidant defense system in the apple snail eggs, the role of ovorubin

A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid-apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid-protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.     

The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation

Background

Fieldwork has thoroughly established that most eggs are intensely predated. Among the few exceptions are the aerial egg clutches from the aquatic snail Pomacea canaliculata which have virtually no predators. Its defenses are advertised by the pigmented ovorubin perivitellin providing a conspicuous reddish coloration. The nature of the defense however, was not clear, except for a screening for defenses that identified a neurotoxic perivitellin with lethal effect on rodents.Ovorubin is a proteinase inhibitor (PI) whose role to protect against pathogens was taken for granted, according to the prevailing assumption. Through biochemical, biophysical and feeding experiments we studied the proteinase inhibitor function of ovorubin in egg defenses.

Methodology/Principal Findings

Mass spectrometry sequencing indicated ovorubin belongs to the Kunitz-type serine proteinase inhibitor family. It specifically binds trypsin as determined by small angle X-ray scattering (SAXS) and cross-linking studies but, in contrast to the classical assumption, it does not prevent bacterial growth. Ovorubin was found extremely resistant to in vitro gastrointestinal proteolysis. Moreover feeding studies showed that ovorubin ingestion diminishes growth rate in rats indicating that this highly stable PI is capable of surviving passage through the gastrointestinal tract in a biologically active form.

Conclusions

To our knowledge, this is the first direct evidence of the interaction of an egg PI with a digestive protease of potential predators, limiting predator''s ability to digest egg nutrients. This role has not been reported in the animal kingdom but it is similar to plant defenses against herbivory. Further, this would be the only defense model with no trade-offs between conspicuousness and noxiousness by encoding into the same molecule both the aposematic warning signal and an antinutritive/antidigestive defense. These defenses, combined with a neurotoxin and probably unpalatable factors would explain the near absence of predators, opening new perspectives in the study of the evolution and ecology of egg defensive strategies.     

Isolation and characterization of a novel perivitellin from the eggs of Pomacea scalaris (Mollusca, Ampullariidae)

Perivitellins are important components of the perivitelline fluid (PVF) that surrounds gastropod embryos. The glyco-lipo-carotenoprotein ovorubin (OR) from eggs of the snail Pomacea canaliculata has been the most studied to date. Here we report the characterization of scalarin (SC), a glyco-lipo-carotenoprotein from the PVF of P. scalaris. SC was purified by ultracentrifugation and exclusion chromatography. It is the major egg protein, representing 64% of the total soluble protein. The particle has a hydration density of 1.26 g/ml, an apparent molecular mass of 380 kDa and it is an elongated compact protein as estimated by small angle X-ray scattering (SAXS). It is composed of three subunits of ca. 35, 28, and 24 kDa noncovalently bonded. SC is highly glycosylated (carbohydrate content 20.1%, by wt.), with a low lipid content (0.7%), being esterified sterols, pigments and polar lipids the most abundant lipid classes. HPTLC and spectrophotometric analysis of the carotenoid fraction revealed the presence of free astaxanthin (ASX; 62.0%), and an unidentified carotenoid (38.0%). The carotenoid-apoprotein interaction was studied by spectrophotometry. Carotenoids do not seem to affect the structural characteristics of the oligomer. However, the carotenoid-protein association protected ASX against oxidation. The cross-reactivity between SC and perivitellins of P. canaliculata was tested using polyclonal antibodies (PAb) against SC, OR, and perivitellin PV2. The PAbs failed to cross-react with any egg proteins of either the same or other species. SC, among other functional similarities with OR, would be an antioxidant carrier, protecting at the same time carotenoids from oxidation in the perivitellin fluid of the egg.     

Structural characterization and low-resolution model of BJ-48, a thrombin-like enzyme from Bothrops jararacussu venom

Thrombin-like enzymes (TLEs) are important components of snake venoms due to their involvement in coagulopathies occurring on envenoming. Structural characterization of this group of serine proteases is of utmost importance for better understanding their unique properties. However, the high carbohydrate content of some members of this group prevents successful crystallization for structural determination. Circumventing this difficulty, the structure of BJ-48, a highly glycosylated TLE from Bothrops jararacussu venom, was studied in solution. At pH 8.0, where the enzyme displays maximum activity, BJ-48 has a radius of gyration (Rg) of 37 A and a maximum dimension (D(max)) of 130 A as measured by small-angle X-ray scattering (SAXS) and a Stokes radius (SR) of 50 A according to dynamic light scattering (DLS) data. At the naturally more acidic pH (6.0) of the B. jararacussu venom BJ-48 behaves as a more compact particle as evidenced by SAXS (R(g)=27.9 A and D(max)=82 A) and DLS (SR=30 A) data. In addition, Kratky plot analysis indicates a rigid shape at pH 8.0 and a flexible shape at pH 6.0. On the other hand, the center of mass of intrinsic fluorescence was not changed while varying pH, possibly indicating the absence of fluorescent amino acids in the regions affected by pH variation. Circular dichroism experiments carried out with BJ-48 indicate a substantially random coiled secondary structure that is not affected by pH. Low-resolution model of BJ-48 presented a prolate elongated shape at pH 8.0 and a U-shape at 6.0. BJ-48 tertiary structure at pH 6.0 was maintained on heating up to 52 degrees C and was completely lost at 75 degrees C. The possible existence of two pH-induced folding states for BJ-48 and its importance for the biological role and stability of this enzyme was discussed.     

Urea-dependent unfolding of HIV-1 protease studied by circular dichroism and small-angle X-ray scattering

HIV-1 protease is responsible for the maturation of infective virions, and is one of the targets of drugs against AIDS. It is an aspartic protease with a 99-resiude polypeptide dimerized. Previous study with fluorescence and sedimentation measurements revealed that the protein was unfolded with concomitant dissociation of the subunits. In the present study, we investigated urea-dependent unfolding of HIV-1 protease with CD and SAXS in order to monitor the secondary structure and the global size and shape of the molecule, respectively. The unfolding parameters estimated by both methods were almost the same, indicating that the dissociation of the subunits accompanied the disruption of their internal structures. This is in line with the previous results, and moreover some residual structures were suggested to be present in the unfolded state. The distinct difference, as compared with the unfolding of pepsin, was interpreted from the point of their molecular architectures.     

Astaxanthin binding and structural stability of the apple snail carotenoprotein ovorubin

Ovorubin (OR) is the major perivitellin of the eggs of Pomacea canaliculata. The astaxanthin (ASX) binding and structural stability of OR were investigated by fluorescence spectroscopy and circular dichroism (CD). The apo-OR (without astaxanthin) shows a single, high affinity binding site for ASX (K(D)=0.5 microM). The quenching of tryptophan fluorescence by ASX indicates that about 22% are near the carotenoid-binding site in a non-polar environment, as indicated by tryptophan resonance energy transfer to the ligand. Secondary structure (alpha+beta) was virtually not affected by cofactor removal. Holo-OR shows unusually high thermal stability. The removal of ASX does not affect the thermal or chemical stability of the quaternary structure. In conclusion, although subtle changes were observed, ASX is not essential for OR stability, unlike most invertebrate carotenoproteins. This supports the idea that OR plays an important physiological role in the storage, transport and protection of carotenoids during snail embryogenesis.     

N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH.

Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs. The difference between the two is the presence of the prosegment in pepsinogen. In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability. No effect on stability was observed for the deletion of a peptide from the C-terminal domain. However, mutations on the N-terminal fragment had a major impact on stability. At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type. The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing. The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability. Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.     

Characterization of the major egg glycolipoproteins from the perivitellin fluid of the apple snail Pomacea canaliculata

Ovorubin and PV2 are the major lipoglycocarotenoproteins present in the perivitellus of the freshwater snail eggs of Pomacea canaliculata, a rapidly expanding rice field pest. We have previously characterized these two particles regarding their lipid and protein compositions, their synthesis and tissular distribution, and their contributions of energy and structural precursors for the developing embryo. In the present study, we have characterized the glycosidic moieties associated to these perivitellines. Both proteins were isolated from egg homogenates by ultracentrifugation, and high performance liquid chromatography (HPLC) using anionic exchange and size exclusion columns. Total carbohydrates accounted for 17.8% and 2.5% (w/w) of the apparent molecular mass of ovorubin and PV2, respectively. Analysis by size exclusion chromatography showed that the amount of O-linked oligosaccharides is higher than that of the N-linked species (59% and 67% w/w of total carbohydrates of ovorubin and PV2, respectively). Glycosylation patterns were determined by a set of biotinilated lectins onto blotted purified proteins. Lectin affinities confirmed the presence of aspargine-linked carbohydrates, probably of hybrid and high mannose types. Jacaline affinity suggested the presence of O-linked residues derived from the T-antigen. Total carbohydrate composition determined by gas liquid chromatography (GLC) showed that mannose was the major monosaccharide in both perivitellins followed by GlcNAc and Gal in ovorubin, and Gal and GlcNAc in PV2. Only one fatty acid (22:1 n-9) accounted for 46% and 56% of the fatty acids present in ovorubin and PV2, respectively. Carbohydrate role on these reserve proteins during embryogenesis of the apple snail is discussed.     

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.     

Metabolism of ovorubin,the major egg lipoprotein from the apple snail

The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad–digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25°C for 12 h with ¹⁴C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad–digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with ³H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.     

Ethanol and acetonitrile induces conformational changes in porcine pepsin at alkaline denatured state

Pepsin, a member of the aspartate protease family, exists in a partially unfolded state at alkaline pH where the N-terminal domain of pepsin has a flexible structure while the C-terminal domain has a highly folded structure. In this work, the conformational stability of porcine pepsin in an alkaline denatured (A(D)) state against acetonitrile and ethanol solvents was studied using a combination of electronic circular dichroism (ECD) and fluorescence techniques. The ECD results demonstrate that both ethanol and acetonitrile induce secondary structural changes in pepsin at A(D) state. However, the minimum concentration required to induce significant secondary structural changes in pepsin varies for ethanol (>30%, v/v) and acetonitrile (>60%, v/v) solvents. At maximum concentration used (90%, v/v), both solvents induce predominantly β-sheet conformation. Unlike acetonitrile, ethanol induces significant amount of non-native α-helical conformations at the intermediate concentrations (50-80%). The tryptophan fluorescence results demonstrate that both acetonitrile and ethanol induce substantial changes in the tertiary structure of pepsin in the A(D) state above certain concentrations. The current results have important implications in understanding the effect of co-solvents on the conformation of proteins in the "denatured state".     

Gastric antibacterial efficiency is different for pepsin A and C

The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.     

Study on the interaction of β‐carotene and astaxanthin with trypsin and pepsin by spectroscopic techniques

β‐Carotene and astaxanthin are two carotenoids with powerful antioxidant properties, but the binding mechanisms of β‐carotene/astaxanthin to proteases remain unclear. In this study, the interaction of these two carotenoids with trypsin and pepsin was investigated using steady‐state and time‐resolved fluorescence measurements, synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The experimental results indicated that the quenching mechanisms of trypsin/pepsin by the two carotenoids are static processes. The binding constants of trypsin and pepsin with these two carotenoids are in the following order: astaxanthin–trypsin > astaxanthin–pepsin > β‐carotene–trypsin > β‐carotene–pepsin, respectively. Thermodynamic investigations revealed that the interaction between the two carotenoids and trypsin/pepsin is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. In addition, as shown by synchronous fluorescence spectroscopy, UV–vis absorption spectroscopy and CD, the two carotenoids may induce conformational and microenvironmental changes in trypsin/pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of the two carotenoids with trypsin/pepsin and is helpful in understanding their effect on protein function and their biological activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Biochemical defects of mutant nudel alleles causing early developmental arrest or dorsalization of the Drosophila embryo

The nudel gene of Drosophila is maternally required both for structural integrity of the egg and for dorsoventral patterning of the embryo. It encodes a structurally modular protein that is secreted by ovarian follicle cells. Genetic and molecular studies have suggested that the Nudel protein is also functionally modular, with a serine protease domain that is specifically required for ventral development. Here we describe biochemical and immunolocalization studies that provide insight into the molecular basis for the distinct phenotypes produced by nudel mutations and for the interactions between these alleles. Mutations causing loss of embryonic dorsoventral polarity result in a failure to activate the protease domain of Nudel. Our analyses support previous findings that catalytic activity of the protease domain is required for dorsoventral patterning and that the Nudel protease is auto-activated and reveal an important role for a region adjacent to the protease domain in Nudel protease function. Mutations causing egg fragility and early embryonic arrest result in a significant decrease in extracellular Nudel protein, due to defects in post-translational processing, stability, or secretion. On the basis of these and other studies of serine proteases, we suggest potential mechanisms for the complementary and antagonistic interactions between the nudel alleles.     

Activation of proenzyme of acidic protease from human seminal plasma.

The kinetics and the extent of the conversion of the proenzyme into the active acidic protease (EC 3.4.23.--) of human seminal plasma were dependent on acidic pH. Between pH 2 and 4, the initial rate of the activation was first-order with respect to the proenzyme. Between pH 4.5 and 5, the rate deviated from the first-order with an initial lag period which can be abolished by adding an excess amount of the acidic protease or pepsin. The extent of the activation was complete between pH 2 and 3 and became incomplete between pH 4 and 5. Addition of the acidic protease or pepsin did not alter the extent of the activation at the high pH values. According to the chromatographic profile on a Sephadex G-75 column, the activation products (namely active acidic protease and an activation peptide) obtained at pH 3 and those obtained at pH 4.5 were identical. The molecular weight of the activation peptide obtained at pH 3 was 6900; its amino acid composition was analyzed and compared with those of the proenzyme and the acidic protease. Remarkable similarity between the amino acid composition of the acidic protease and that of human pepsin was observed. In the presence of an excess amount of hemoglobin, the conversion of the proenzyme was self-activated and showed an initial lag period. Addition of acidic protease did not change the rate of self activation or the lag period.     

Engineered single-domain antibodies with high protease resistance and thermal stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (V(H)Hs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant V(H)Hs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant V(H)H pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the V(H)Hs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.     

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.     

Special Features of the Three-Dimensional Structure Defining Properties of Aspartic Proteases

A brief account is given of the specific interactions of some amino acid residues in aspartic proteases of both higher organisms and retroviruses that determine important protease properties: the anomalously low isoelectric point of pepsin and its stability at pH close to 1; the ability of one of the carboxyl groups in the active site of proteases of higher organisms to retain a charged state at any pH value; and the protonated state of another carboxyl, which is necessary for enzymatic activity. It is also explained how such states can be induced in retroviral proteases.