Immobilization of polyribonucleotides in agarose gels at high ionic strength

Purine polyribonucleotides poly(A), poly(G), and poly(I) associate reversibly with agarose gels at high NaCl molarities over the pH range 6–10, at 20°?40°C. Pyrimidine polyribonucleotides poly (C) and poly(U) could not be immobilized in agarose gels under the above conditions. However, poly(C) could be immobilized in agarose without precipitation between pH 3.2 and 4.0. Association of poly(G) and poly(I) with agarose appears to decrease progressively with deprotonation of their purine residues, and both polymers interact with the gel very weakly above pH 10 regardless of NaCl concentration. The binding to agarose of these polymers at pH 7.5 is also strongly influenced by temperature in the range 20°?40°C. The association of single-stranded poly(A) is only shifted toward higher NaCl molarities by increased pH; its binding is also little affected by temperature in the above range. At NaCl molarities effecting the saturating retention in agarose and at neutral pH, the immobilization of several polynucleotides could be prevented by urea in a concentration-dependent manner. The corresponding profiles of urea molarity appear to disclose a number of hydrophobic interactions between polynucleotides and agarose, some of which could be relatively strong, especially in the case of poly(A).     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Multinuclear magnetic resonance studies of collagen molecular structure and dynamics

We have measured the percentages of cis and trans Gly-Pro and X-Hyp peptide bonds in thermally unfolded type I collagen. ¹³C-nmr solution spectra show that 16% of the Gly-Pro and 8% of the X-Hyp bonds are cis in unfolded chick calvaria collagen. These results support the hypothesis that cis–trans isomerization is that rate-limiting step in the propagation of the collagen triple helix. We have used multinuclear solid-state nmr to study the molecular dynamics of the collagen backbone in tendon, demineralized bone, and intact bone as a function of temperature, hydration, and pH. These studies show that collagen backbone motions are characterized by a broad distribution of correlation times, τ, covering the range from 10^?4 to 10^?9 s. In the case of nonmineralized collagen, the root-mean-square fluctuations in azimuthal angle, γ_rms, range from ca. 10° when τ ～ 10^?9 s to ca. 30° when τ < 10^?4 s; in the case of bone collagen, γ_rms values are about half as large as those found in nonmineralized collagen. Backbone motions are negligible at temperatures below ?25°C. This is also the case at 22°C when demineralized bone collagen is lyophilized. In contrast, flexibility of hydrated demineralized bone collagen greatly increases as pH is lowered from 7 to 2. The more limited flexibility observed at neutral pH is a consequence of the intermolecular interactions that contribute to fibril organization and strength. However, the fibrils retain significant flexibility at physiological pH, enabling them to distribute stress and dissipate mechanical energy.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Interactions between collagen chains and fiber formation

The temperature-dependent dissociation of neutral salt-soluble collagen into its component chains was measured in 0.6–1.6 M urea solutions at pH 7.3. The temperature-dependent association of the same radiocactively labeled collagen into fibers was measured in 0–0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39°C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30°C. We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.     

Reversibility of thermal transitions in proteins: Racemization and aggregation as factors in the reversible denaturation of a soluble keratin derivative (SCMKA)

The reversibility of the thermal denaturation of a low-sulfur fraction of solubilized wool keratin (SCMKA) has been studied under a variety of conditions of time, protein concentration, and pH. Two types of irreversibility for the transition have been encountered. One of these is associated with an aggregation of the protein on denaturation to give a product which may contain elements of a β conformation. This type of irreversibility is favored by high protein concentration, and the original conformation may in fact be regained if the aggregated structure is broken down by a solvent such as 8M urea and the urea subsequently removed by dialysis. The other type of irreversibility appears to be due to racemization of the protein. It does not seem to be dependent on protein concentration and is apparent only at temperatures beyond the actual transition range (～40–65°C) at pH values below 11, At pH 12, however, racemization appears to proceed slowly even at 4°C. The thermal transition at pH 9 and pH 10 has been shown to be multistage in nature. Over the pH range 9–12 there is a progressive decrease in thermal stability with increase of pH. Addition of NaCl at pH 10 leads to an increase in thermal stability of the molecule.     

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

Molecular basis of cooperativity in protein folding. V. Thermodynamic and structural conditions for the stabilization of compact denatured states

The heat-denatured state of proteins has been usually assumed to be a fully hydrated random coil. It is now evident that under certain solvent conditions or after chemical or genetic modifications, the protein molecule may exhibit a hydrophobic core and residual secondary structure after thermal denaturation. This state of the protein has been called the “compact denatured” or “molten globule” state. Recently is has been shown that α-lactalbumin at pH < 5 denatures into a molten globule state upon increasing the temperature (Griko, Y., Freire, E., Privalov, P. L. Biochemistry 33:1889–1899, 1994). This state has a lower heat capacity and a higher enthalpy at low temperatures than the unfolded state. At those temperatures the stabilization of the molten globule state is of an entropic origin since the enthalpy contributes unfavorably to the Gibbs free energy. Since the molten globule is more structured than the unfolded state and, therefore, is expected to have a lower configurational entropy, the net entropic gain must originate primarily from solvent related entropy arising from the hydrophobic effect, and to a lesser extent from protonation or electrostatic effects. In this work, we have examined a large ensemble of partly folded states derived from the native structure of α-lactalbumin in order to identify those states that satisfy the energetic criteria of the molten globule. It was found that only few states satisfied the experimental constraints and that, furthermore, those states were part of the same structural family. In particular, the regions corresponding to the A, B, and C helices were found to be folded, while the β sheet and the D helix were found to be unfolded. At temperatures below 45°C the states exhibiting those structural characteristics are enthalpically higher than the unfolded state in agreement with the experimental data. Interestingly, those states have a heat capacity close to that observed for the acid pH compact denatured state of α-lactalbumin [980 cal (mol.K)^?l]. In addition, the folded regions of these states include those residues found to be highly protected by NMR hydrogen exchange experiments. This work represents an initial attempt to model the structural origin of the thermodynamic properties of partly folded states. The results suggest a number of structural features that are consistent with experimental data. © 1994 Wiley-Liss, Inc.     

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.     

Preparation of extracts from mature spruce needles for enzymatic analyses

It was possible to extract simultaneously several active enzymes involved in the carbohydrate or the amino acid metabolism from spruce needles [ Picea abies (L.) Karst.] when a) a 100 m M Na-Pi buffer of pH 7.5 containing 5% PVPP and 0.5% Triton X-100 was used and when b) the resulting crude extracts were freed from lowmolecular-weight compounds by gel-chromatography using the separation medium Fractogel TSK HW-40. Besides Triton X-100, Triton X-305, Myrij-52 and Brij-35 were tested, but 0.5% Triton X-100 brought about the most active enzyme extracts. In crude extracts prepared from spruce needles during the early summer a high increase in absorbance at 334 nm was observed when the co-substrate NADP⁺ was added, thus making reliable spectrophotometric assays impossible. The interfering low-molecular-weight substances could be eliminated by gel chromatography. As separation media Bio-Gel P-6 DG, Sephadex G-25 m, Trisacryl GF 05 and Fractogel TSK HW-40 (F) were tested, with Fractogel yielding the highest activities.
With the methods described in this paper the activities of the following enzymes were determined: glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate isomerase (EC 5.3.1.9), shikimate dehydrogenase (EC 1.1.1.25), NAD⁺-malate dehydrogenase (EC 1.1.1.37), glutamate dehydrogenase (EC 1.4.1.2), aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2). The activities estimated for NAD⁺-malate dehydrogenase and 6-phosphogluconate dehydrogenase are in the range of those published for the needle enzymes of white spruce and Scots pine, respectively.     

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

USE OF ULTRASONIC SPECTROSCOPY AND VISCOSIMETRY FOR THE CHARACTERIZATION OF CHICKEN SKIN COLLAGEN IN COMPARISON WITH COLLAGENS FROM OTHER ANIMAL TISSUES

Use of traditional sources of collagen such as pork, bovine, and carp has some limitations. Chicken skin can be valuable alternative. In this work collagen was isolated from chicken skin using a modified procedure. Molecular properties of chicken collagen were analyzed and compared to collagen from other animal skins. Acid-soluble collagen type I was obtained with a yield of 25% and water content around 67%. Viscosimetry and ultrasonic spectroscopy were newly used for molecular characterization. By ultrasonic attenuation measurements, a pre-aggregation phase in the interval from 20°C to 27°C was observed, which is a proof of disaggregation and liquefaction. From 40°C upward, the liquefaction process finishes and aggregation continues. In a bovine sample this phenomenon starts at 40°C, in chicken at 50°C, and continues until 70°C. By viscosimetry, the denaturation temperature was confirmed as 40°C for bovine and 50°C for chicken collagen. Chicken collagen has a two times higher lysine level than bovine, which provides molecular stability side-chain interactions. With regard to higher thermal stability and favorable amino acid composition, waste chicken skin has the potential to be an excellent alternative source of raw collagen with applications in the food industry and biomedicine.     

Collagen fibril formation in vitro at nearly physiological temperatures

Fibril formation by collagen from piglet skin was studied at temperatures of 28–39°C. Collagen fibrils obtained in this temperature range differ in the degree of ordering. Electron microscopy shows that fibrils of minimal diameter are formed at physiological pH, ionic strength (PBS), and temperature. The greater diameter of fibrils formed at 34.5°C is due to enhanced collagen hydration. Fibril diameter at 38.5°C is increased because of cooperative unfolding of the triple helix and weaker binding between collagen molecules. The optimal temperature for fibrillogenesis appears to be 36.5°C, and such fibrils are most similar to those observed in vivo.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.

At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I₃, is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction (k = 0.7 × 10⁶m^?1s^?1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP ² binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper).I₃ shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I₃ and by tyrosine absorbance.The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition.     

The low-pH unfolded state of the C-terminal domain of the ribosomal protein L9 contains significant secondary structure in the absence of denaturant but is no more compact than the low-pH urea unfolded state

There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Complete conformational stability of kinetically stable dimeric serine protease milin against pH,temperature, urea,and proteolysis

Spectroscopic, calorimetric, and proteolytic methods were utilized to evaluate the stability of the kinetically stable, differentially glycosylated, dimeric serine protease milin as a function of pH (1.0–11.0), temperature, urea, and GuHCl denaturation in presence of 8 M urea at pH 2.0. The stability of milin remains equivalent to that of native at pH 1.0–11.0. However, negligible and reversible alteration in structure upon temperature transition has been observed at pH 2.0 and with 1.6 M GuHCl. Irreversible and incomplete calorimetric transition with apparent T _m > 100°C was observed at basic pH (9.0 and 10.0). Urea-induced unfolding at pH 4.0, and at pH 2.0 with GuHCl, in presence of 8 M urea also reveals incomplete unfolding. Milin has been found to exhibit proteolytic resistant in either native or denatured state against various commercial proteases. These results imply that the high conformational stability of milin against various denaturating conditions enable its potential use in protease-based industries.