Photocontrol of affinity chromatography. I. Trypsin purification by photosensitive soybean trypsin inhibitor (STI) gel

Soybean trypsin inhibitor (STI) was immobilized on the agarose gel modified with spiropyran compound (spiropyran gel), and photocontrolled binding and releasing of trypsin was examined. The STI-spiropyran gel showed reverse photochromism. Trypsin was bound on the STI-spiropyran gel in the dark and released with visible light irradiation. The optimum conditions for photocontrolled binding and releasing of trypsin were pH 6.6 and the buffer concentration of 0.05 m. Approximately 60–80% of bound trypsin was released with visible light irradiation. The activity of released trypsin was the same as that of native trypsin. Approximately 21-fold purification of trypsin was performed with the STI-spiropyran gel column.     

Photocontrol of affinity chromatography: Purification of asparaginase by photosensitive AHA-gel

An agarose gel modified with N-(ω-aminohexytl)-L -aspartic acid (AHA) and spiropyran compound (AHA–spiropyran gel) was prepared and the photocontrolled binding and releasing of asparaginase were investigated with the AHA–spiropyran gel. Asparaginase was bound on the AHA–spiropyran gel under visible light and was released in the dark. The optimum conditions for photocontrolled binding and releasing of asparaginase were a 0.05M phosphate buffer concentration and pH 7.0. Seventy-five percent of the bound asparaginase was released from the AHA–spiropyran gel column in the dark. Ninetyfold purification of asparaginase was performed with the AHA–spiropyran gel Column.     

Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.     

Amino acid esterification by alpha-chymotrypsin immobilized in spiropyran membrane.

α-Chymotrypsin was immobilized in a collagen membrane modified with a spiropyran compound. The immobilized chymotrypsin was used for the esterification of N-acetyl-l-tyrosine (AT). N-Acetyl-l-tyrosine ethyl ester (ATEE) was synthesized from AT and ethanol by immobilized chymotrypsin under visible light. The optimum pH for the esterification was 7. An increase of the chymotrypsin content in the spiropyran-collagen membrane increased the rate and the yield of ATEE. The yield of ATEE reached 40% under visible light. Initially, ATEE was synthesized in the dark. However, the ATEE synthesized was gradually hydrolyzed in the dark. The amount of ATEE in the reaction mixture increased with irradiation by visible light and decreased in the dark. Therefore, the esterification of N-acetyl-l-tyrosine was controlled by light irradiation.     

Photo control of enzyme activity of alpha-amylase.

Photo control of enzyme activity was performed by attaching a photochromic spiropyran compound to α-amylase. Modified α-amylase exhibited reverse photochromism in water: a colored form in the dark and a colorless form under light irradiation, which indicated that bound spiropyran possessed a hydrophilic structure (an open-ring form) in the dark and a hydrophobic structure (a closed-ring form) under light irradiation. The activity of modified α-amylase under light irradiation was extremely retarded as compared with that determined in the dark. The photo-induced change of the activity reversibly occurred in accordance with the photochromism of bound spiropyran. The mechanism of the photo control is discussed.     

Effect of riboflavin and light on the secondary structure of DNA

S1 nuclease hydrolysis and benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography have been used to study the effect of riboflavin and visible light on DNA. Native calf thymus DNA was incubated with riboflavin in the presence of fluorescent light for various time periods and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Incubation with riboflavin and illumination caused adherence to BND-cellulose indicating the production of single stranded regions or breaks in the native double stranded molecules. However, when incubation was done in dark and in the presence of triplet excited state quencher, potassium iodide, a reduced adherence of DNA to BND-cellulose was seen. Plasmid pBR322 DNA was also treated with riboflavin under these conditions and subjected to agarose gel electrophoresis. No degradation could be seen in dark incubated and potassium iodide treated samples. These results indicate that the adherence of DNA to BND-cellulose in dark is possibly due to the binding of aromatic residues to the resin suggesting the formation of a complex between riboflavin and DNA.     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Modifications of Δ5-3-ketosteroid isomerase induced by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent Δ6-testosterone agarose

The nature of the products formed during the photoinactivation of Δ⁵-3-ketosteroid isomerase in the presence of the solid-phase photoaffinity reagent Δ⁶-testosterone succinyl agarose has been investigated after ultraviolet irradiation. The polypeptide products eluted from the agarose phase by sodium cholate, sodium dodecyl sulfate, and pH 10.5 triethylamine buffer have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, pH 4–6 gel isoelectric focusing, and amino acid analysis. The amino acid compositions of the cholate eluted and SDS eluted products are found to be similar to that of native isomerase, whereas the covalently bound polypeptide eluted by pH 10.5 triethylamine possesses a distinetly different composition. Digestion of the covalently bonded isomerase polypeptide with trypsin yields an agarose-bound peptide fraction that has been characterized by its amino acid composition. This composition is different from that of the undigested covalently bound polypeptide and suggests that the site of covalent attachment lies somewhere between residues 28 and 45 of the isomerase polypeptide.     

Isolation and immunochemical studies of dog trypsin

Dog trypsin (EC 3.4.4.4) was isolated from dog pancreatic juice on SP-Sephadex C-50. The preparation was homogeneous on disc electrophoresis at pH 4.3. On agarose gel electrophoresis at pH 8.6, dog pancreas trypsinogen had the mobility of an alpha 2-globulin and trypsin the mobility of a beta-globulin. On gel filtration on Sephadex G-75 at pH 4.0, dog trypsin was eluted in the same fractions as bovine trypsin. It was inhibited by soybean trypsin inhibitor. Rabbit anti-dog trypsin inhibited the caseinolytic activity of bovine trypsin by about 60%.     

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-beta-glucoside.

The structural stability of bacteriorhodopsin (bR) solubilized by octyl-beta-glucoside was studied by measuring the denaturation kinetics under visible light irradiation and in the dark. The denaturation of bR solubilized by 50 mM octyl-beta-glucoside was very slow at room temperature when it was left in the dark. However, its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation and in the dark could be well described by a single decay constant. The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light irradiation was 12.5 kcal/mol, which was much smaller than the corresponding value in the dark, 26.2 kcal/mol. These results strongly suggest that some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D crystalline state. Comparing the denaturation behavior in the 2D crystalline state and that in the octyl-beta-glucoside-solubilized state, our findings suggest that protein-protein interaction contributes to the stability of this protein.     

Stabilization of retinol through incorporation into liposomes

Chemical and photochemical processes during storage and preparation rapidly degrade retinol, the most active form of vitamin A. Therefore, the efficacy of incorporation into liposomes in order to modulate the kinetics of retinol degradation was investigated. Retinol was readily incorporated into multilamellar liposomes that were prepared from soybean phosphatidylcholine; the extent of the incorporation was 98.14 +/- 0.93% at pH 9.0 at a ratio of 0.01 : 1 (wt : wt) retinol : phospholipid. It was only marginally lower at higher retinol concentrations. The pH of the hydration buffer had a small effect. The incorporation efficiency ranged from 99.25 +/- 0.47% at pH 3 to 97.45 +/- 1.13% at pH 11. The time course of the retinol degradation in the aqueous solution in liposomes was compared to that of free retinol and free retinol with alpha-tocopherol under a variety of conditions of pH (3, 7, and 11), temperature (4, 25, 37, and 50 degrees ), and light exposure (dark, visible, and UV). The retinol that was incorporated into the liposomes degraded significantly slower than the free retinol or retinol with alpha-tocopherol at pH 7 and 11. At pH 3, where the free retinol degrades rapidly, the degradation kinetics were similar in liposomes and the presence of alpha-tocopherol. At pH 7.0 and 4 degrees in the light, for example, free aqueous retinol was completely degraded within 2 days, while only 20% of the retinol in the liposomes were degraded after 8 days. In general, the protective effect of the liposome incorporation was greater at low temperatures, at neutral and high pH, and in the dark. The results suggest that protection is greater in the solid, gel phase than in the fluid liquid crystalline phase lipids. These results indicate that the incorporation into liposomes can extend the shelf-life of retinol under a variety of conditions of temperature, pH, and ambient light conditions.     

Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase

3-Hydroxy-3-methylglutaryl-CoA lyase has been purified to homogeneity from avian liver mitochondria. Affinity chromatography of a partially purified preparation on agarose hexane 3',5'-ADP produces enzyme of high specific activity (351 units/mg). A total purification of 1750-fold over the mitochondrial matrix fraction is achieved. The purified enzyme is stable when stored in 30% glycerol with millimolar levels of dithiothreitol. Divalent cations (e.g. Mg2+, Mn2+) and thiol-protecting agents stimulate enzyme activity under assay conditions. The enzyme binds hydroxymethylglutaryl-CoA with a Km = 8 microM. Optimal enzyme activity, measured at pH = 8.9, is 7-fold higher than activity at physiological pH. The apparent molecular weight of the native enzyme, estimated by gel filtration on Sephadex G-100, is approximately 49,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a dimer, composed of 27,000-dalton subunits. Assuming one active site per subunit, a turnover number of 158 s-1 (pH 8.2; 30 degrees C) is calculated. Antibodies have been prepared against homogeneous hydroxymethylglutaryl-CoA lyase. Ouchterlony double diffusion patterns verify the homogeneity of the preparation. Incubation of enzyme with antiserum results in virtually complete inhibition of enzyme activity.     

Visible light during mycelial growth and conidiation of Metarhizium robertsii produces conidia with increased stress tolerance

Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m(-2)). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.     

Preparation and characterization of an enzymatically active immobilized derivative of myosin.

Purified skeletal muscle myosin (EC 3.6.1.3) has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.     

Production and characterization of glutathione-S-transferase fused with a poly-histidine tag

Schistosoma japonicum glutathione-S-transferase (SjGST) was genetically engineered with a poly-histidine tag at the C-terminus and highly expressed in Escherichia coli. Both SjGST and the tagged protein, SjGST/His, were purified with glutathione Sepharose 4B gels and subsequently studied for their activities, antibody-binding abilities, and metal affinities. The production level of active SjGST/His was higher than that of SjGST. Both proteins had similar specific catalytic activities and binding abilities with anti-SjGST antibody, while the antibody against poly-histidine recognized only SjGST/His. Proteolytic degradation was occasionally observed in aged dialyzed SjGST/His preparation. Under a native condition, the Co(2+)-chelated TANOL gel (Co-TANOL) had a better binding specificity to the tagged protein than did the Ni(2+)-chelated nitriloacetic acid (Ni-NTA) agarose gel. However, the binding capacity of the Ni-NTA gel for SjGST/His was 2-fold higher than that of the Co-TANOL one. To increase the native binding specificity of the Ni-NTA gel, 20 mM imidazole had to be added to the washing solution. In a denatured state, both gels could only capture SjGST/His, and the binding capacity of the Ni-NTA gel was nearly 2-fold higher than that of the Co-TANOL gel. The binding association constants of both gels with SjGST/His did not differ greatly under either condition. The study demonstrated that the C-terminal addition of the poly-histidine tag to SjGST increased the metal affinity of the enzyme to the Co-TANOL gel under both native and denaturing conditions and to the Ni-NTA gel under denaturing conditions, whereas the enzymatic activity and antibody-binding ability were not affected.     

Operational stability of copolymerized enzymes at elevated temperatures

The copolymerization method of immobilization was used to obtain preparations of enzymes covalently incorporated in polyacrylamide gel. They possess properties making them suitable for practical use. First, the preparations are hundreds of times more stable against irreversible thermoinactivation than native enzymes. Second, on immobilization, the reversible conformational changes which also lower enzyme activity at elevated temperatures are completely suppressed. As a result, the temperatures of maximum activity for trypsin and alpha-chymotrypsin covalently entrapped in polyacrylamide gel are 75 and 70 degrees C, respectively-25 and 30 degrees C higher than the corresponding values for the native enzymes. Therefore, the copolymerized enzyme preparations have a high operational stability at elevated temperatures.     

The Effect of organic nitrogen sources on recombinant glucoamylase production by Aspergillus niger in chemostat culture

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 +/- 0.01 h(-1), pH 5.4). In cultures supplemented with l-alanine, l-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA.     

Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii

Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed.     

Enzymatic liquefaction of agarose above the sol–gel transition temperature using a thermostable endo-type β-agarase,Aga16B

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of d-galactose and 3,6-anhydro-l-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol–gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol–gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol–gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40^T, was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol–gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol–gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.