Adsorption Of Trypsin Research Articles

Use ofp-nitrophenyl chloroformate chemistry to immobilize protein on orthopedic biomaterials

Biochemical surface modification involves covalently immobilizing biomolecules onto biomaterial surfaces to induce specific biological responses. This approach may be useful for enhancing the fixation of orthopedic implants. p-Nitrophenyl chloroformate (p-NPC) was used to immobilize protein on bulk samples of Co-Cr-Mo and Ti-6Al-4V. Activation of both materials was dependent on the concentration of p-NPC, with a maximum of approximately 1.5 active groups/nm2 of nominal surface area. Trypsin was used as a model protein because much is known about its structure and mode of action. Derivatization with 0.65 mg p-NPC/cm2 resulted in significantly greater enzymatic activity (7.4 BAEE [N-(alpha)-benzoyl-L-arginine ethyl ester hydrochloride] units) on the Co-Cr-Mo samples compared with higher concentrations of p-NPC (5 BAEE units) and with simple adsorption of trypsin (1.5 BAEE units). An activity of 10.5 BAEE units was measured on both adsorbed and p-NPC-activated Ti-6Al-4V, with the exception of samples derivatized with 1.95 mg p-NPC/cm2, on which activity was significantly lower (4 BAEE units). In probing the linkages between trypsin and biomaterial by treatment with chaotropic agents, guanidine hydrochloride (GuHCl) was observed to eliminate more enzymatic activity than was urea. On Co-Cr-Mo samples, GuHCl removed nearly all the trypsin activity, while urea significantly decreased the activity only at a concentration of 0.65 mg p-NPC/cm2. Treatment of Ti-6Al-4V samples with GuHCl caused a trend of decreasing activity with increasing concentration of p-NPC, whereas urea had no effect on immobilized trypsin activity.

An affinity adsorbent derived from aminopropyl silica for serine protease chromatography

AbstractA silica‐based adsorbent for affinity chromatography of serine protease was prepared by bonding p‐aminobenzamidine (pABZ) to aminopropyl silica. Silanization of silica with both γ‐aminopropyltrimethoxysilane and γ‐amino‐propyltriethoxysilane under anhydrous conditions led to a monolayer density of primary amino groups. The aminopropyl silica was converted to primary hydroxylcontaining silica via diazotization, and consequently activated with p‐nitrophenyl chloroformate and immobilized with ligand pABZ. The resultant silica–pABZ has a ligand density of 13.8 μmol g−1. Pyridine was indicated by the data to increase the reactivity of the chloroformate toward the hydroxyl group more efficiently than 4‐dimethylaminopyridine. Two stages of adsorption were found in batch adsorption of trypsin with an equilibrium adsorption isotherm of the Langmuir type. When the chromatographic column packed with this silica–p ABZ was operated under a higher flow rate (2·33 cm3 min−1) and with 3 g dm−3 of crude urokinase as the influence, the yield was 55%. Both the flow rate and the concentration of the crude protein were shown, by measuring the dynamic binding capacity from chromatographic experiments, to be the factors which influenced the chromatographic efficiency.

トリプシン共存におけるハイドロタルサイト様化合物へのリン酸イオン交換の挙動

A Cl(-)-intercalated hydrotalcite-like compound (HTAL) is a promising material as a better phosphate adsorbent for further clinical use than the currently marketed aluminum hydroxide gels. In the preceding study, we found that Cl- ions in the interlayer of HTAL can be exchanged with phosphate ions equivalently. In the present study, we have investigated the adsorption of porcine pancreas trypsin on HTAL and the influence of trypsin adsorption on the phosphate/Cl- ion-exchange properties of HTAL. The isotherm for trypsin adsorption at 310K and pH 8.4 showed the curve of BDDT V type and gave an adsorption capacity of 88.6 mg/g for trypsin. The zeta-potential analysis of HTAL suggested that the trypsin adsorption on HTAL at pH 8.4 was restrained by the electrostatic repulsion between HTAL surface and trypsin with positive charge. XRD and nitrogen adsorption studies showed that trypsin was adsorbed on the external surface of HTAL by occupying the area (ca. 22 nm2) per trypsin molecule. The phosphate/Cl- ion-exchange reaction with HTAL was not affected by the presence of trypsin; the phosphate ion-exchange capacity reached 73.6 mg P/g from the solution containing 3200 mg trypsin/dm3. On the other hand, the trypsin adsorption was significantly depressed by the loading of phosphate ions. The XRD analysis of the phosphate-loaded HTAL revealed that the layered structure of hydrotalcite holds after the phosphate ion-exchange, but shows a slight increase of the interlayer distance from 0.3 nm to 0.5 nm by the intercalation of phosphate ions.

A method for the continuous purification of proteins by affinity adsorption

A method was conceived for the purification of biomolecules on a continuous-flow basis using affinity adsorption. The affinity ligand was bound to a nylon belt which was passed sequentially through four chambers to which flows of feedstock, wash medium, eluent and regeneration medium were applied. The target compound was thus removed from the feedstock stream and desorbed into a continuous flow of eluent. Prototype laboratory-scale apparatus was designed and constructed and the technical feasibility of this method was demonstrated using soybean trypsin inhibitor as a ligand for the adsorption of trypsin. The effects of various operational parameters on apparatus function were investigated using this model system. In addition, continuous removal of trypsin from a bovine pancreatic extract was carried out during an 8 h experiment.

Adsorption of enzymes at the solid-liquid interface: I. Trypsin on polystyrene latex

The enzyme, trypsin, has been used to study conformational changes which occur when protein adsorption onto well-characterized, emulsifier-free, polystyrene latex surface takes place. The adsorption isotherm is of the high affinity, Langmuirian type with plateau adsorption of trypsin of 2.8 mg m −2. The enzymic activity of adsorbed trypsin to low molecular weight substrate is found to decrease as the surface coverage decreases indicating that ‘spreading’ or unfolding of the native protein conformation, with consequent loss of enzymic activity, occurs. On the close packed surface such ‘spreading’ is inhibited by steric factors. The view that protein adsorption onto hydrophobic surfaces is dominated by the entropy gain due to protein unfolding to maximize hydrophobic interactions is thus supported.

Adsorption of trypsin onto poly(2‐hydroxyethyl methacrylate)/polystyrene composite microspheres and its enzymatic activity

AbstractPoly(2‐hydroxyethyl methacrylate)/polystyrene (PHEMA/PS) composite microspheres were produced by emulsifier‐free seeded emulsion polymerization for styrene in the presence of PHEMA seed particles. Effects of the surface characteristics of the PHEMA/PS composite microspheres on the adsorption immobilization of trypsin and on its enzymatic activity were discussed. Above 5 mol% of HEMA content, trypsin molecules adsorbed had high activity, 65–100% of the activity of free trypsin. The excellence of the composite microspheres as a carrier for trypsin seems to be closely related with the surface heterogeneity consisting of both hydrophilic and hydrophobic parts.

Adsorption of trypsin onto styrene-2-hydroxyethyl methacrylate copolymer microspheres and its enzymatic activity

Effects of the surface hydrophilicity of styrene-2-hydroxyethyl methacrylate (S-HEMA) copolymer microspheres on the adsorption immobilization of trypsin and on its enzymatic activity were discussed. In the case of a polystyrene (PS) microsphere, the amount of adsorption was large, whereas the enzymatic activity of the adsorbed trypsin was low. On the other hand, in the case of an S-HEMA copolymer microsphere whose HEMA content was 10 mole %, the maximum specific activity of trypsin adsorbed was nearly equal to that of free trypsin, although the amount of adsorption decreased to about 65 % of that in PS. Accordingly, total activity, which is the product of the amount of adsorption and the specific activity, could be enhanced by controlling the surface hydrophilicity of the microsphere.

Effect of Dietary Fiber on Pancreatic Enzyme Activity In Vitro

The importance of various factors for dietary fiber effects on pancreatic enzyme activities was studied in vitro. Pectin of high methylic esterification and guar gum, which reduced enzyme activities, particularly lipase activity, increased the viscosity of duodenal juice. Increasing duodenal juice viscosity by polyethlene glycol also caused a reduction particularly of lipase activity. Pectin of low methylic esterification, which reduced especially amylase and lipase activities, significantly lowered duodenal juice pH. Lowering duodenal juice pH by hydrochloric acid caused a reduction especially of amylase and lipase activities. Pretreating fiber at acid pH tended to increase its enzyme-inhibiting properties. Trypsin adsorption to wheat bran was partly reversible by buffer washings. Changing ionic strength and incubation time appeared relatively unimportant for the effects of the fiber on enzymes. It is assumed that the inhibitory effects of the fiber on enzymes are attributed inter alia to effects on viscosity, pH, and adsorption, and further that gastric acidification of fiber and conditions lowering intestinal pH may enhance these effects.

Performance of affinity chromatography columns

AbstractPerformance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B‐soybean trypsin inhibitor column and a Sepharose 4B‐arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break‐through curves of trypsin, and elution profiles bed height of Sepharose 4B‐STI column was estimated based on these results.

Trypsin Inactivation by Intact Hymenolepis diminuta

Using azoalbumin as a substrate, it was demonstrated that intact Hymenolepis diminuta inactivate trypsin when incubated with this enzyme. At a given enzyme concentration ([E]), the per cent inactivation (%I) was relatively constant as the substrate concentration ([S]) was varied. However, at a given [S], the %I increased as the [E] decreased. It was also demonstrated that inactivation was dependent upon the time worms were exposed to the enzyme, the total worm weight present in the assay, and the pH of the assay medium. The %I was not related to worm surface area, and was unaffected by polyions and calcium ions in the incubation medium. These results are discussed in relation to the possible mechanism and physiological significance of this phenomenon. The mucosal surface of the mammalian intestine and the outer surface of the syncytial epidermis of the cestode, Hymenolepis diminuta, contain intrinsic membrane-bound enzymes (Crane, 1962; Newey et al., 1963; Arme and Read, 1970; Dike and Read, 1971a). Both surfaces are reported to adsorb enzymes of pancreatic origin (Ugolev, 1965; DeLaey, 1966a; Goldberg et al., 1968, 1969; Read, 1972). According to DeLaey (1966b), adsorption of amylase involves interaction with the mucosal glycocalyx and is readily reversible (Jesuitova et al., 1964). Amylase adsorption to the surface of H. diminuta is also reversible and is partially blocked by polyions (Read, 1972). However, the adsorption of proteolytic enzymes to isolated mucosal cells or subcellular particles is not readily reversible (Goldberg et al., 1968, 1969). According to Ugolev (1965), the adsorption of amylase on the mammalian mucosa results in an increase in enzyme activity. However, doubt has been cast on Ugolev's interpretation with the demonstration that the mammalian mucosa has intrinsic membrane-bound amylases (McMichael and Dahlqvist, 1968; Alpers and Solin, 1970). On the other hand, while Hymenolepis does not exhibit intrinsic amylase or disaccharidase activity, adsorption of pancreatic amylase to the worm surface results in an increase in amylolytic activity (Taylor and Thomas, 1968; Read, 1972). Adsorption of trypsin and chymotrypsin to isolated human Received for publication 24 February 1972. * This study was supported by a grant (AI-01384) from the NIH, U. S. Public Health Service. t U. S. Public Health Service Postdoctoral Fellow, 1-F02-AI-45108-01. mucosal cells or subcellular particles does not ter the kinetic parameters of these enzymes (Goldberg et al., 1969). However, in the presence of intact mammalian mucosa, proteolytic enzymes of pancreatic origin are inctivated (Borgstrom et al., 1957; Goldberg et al., 1968, 1969; Pappas and Gallogly, unpublished). Reichenbach-Klinke and Reichenbach-Klinke (1970) reported that trypsin is inactivated in the presence of the tapeworm, Proteocephalus longicollis. The present investigation was concerned with the effects of the tapeworm, Hymenolepis diminuta, on pancreatic trypsin. MATERIALS AND METHODS Hymenolepis diminuta were reared in the beetle, Tenebrio molitor. Male Sprague-Dawley rats (Holtzman Co.) weighing 80 to 100 g were infected with 30 H. diminuta cysticercoids and the worms removed 11 days postinfection. Worms were rinsed in 3 changes of Krebs-Ringer solution containing 25 mM Tris(hydroxymethyl) aminomethane-maleate buffer (pH 7.2) (KRT of Read, Rothman, and Simmons, 1963), randomized into groups (usually 10 worms/group), and incubated in fresh KRT at 37 C for 15 min prior to their addition to the assay medium. The assay medium consisted of 4 ml of KRT and 1 ml of an enzyme solution (Trypsin-type XI: DCC treated. 8,000 BAEE units/mg. Sigma Chemical Co., St. Louis) maintained at 37 C in a shaking water bath. When groups of worms were to be used during assays they were removed from the previous incubation medium (KRT) and allowed to incubate in the KRT-enzyme solution for 15 min prior to the addition of substrate (this period in the KRT-enzyme solution is hereafter referred to as the preincubation period); after preincubation the worms were removed. The reaction was initiated by the addition of 1 ml of an azoalbumin (bovine origin, Sigma) solution prewarmed to 37 C, and allowed to continue for a prede-

Tissue dissociation with proteolytic enzymes: Adsorption and activity of enzymes at the cell surface

Proteolytic enzymes used to dissociate kidney tissue have been shown to adsorb to cell surfaces and persist in an active form for as long as tweny-four hours afterwards. The adsorption and activity of pronase and trypsin at the surfaces of freshly dispersed dog kidney cells was demonstrated by ellipsometry. The effect of various cell washing procedures on desorption of the enzymes and the inhibitory effects of serum on enzyme activity were investigated. Proteolytic enzymes adsorbed to the cell surface were found to prevent the formation of the glycoprotein cell coat material at the surface and so interfere with the attachment, spreading and growth of cells on glass. The possible mechanisms underlying the cell injury and death accompanying enzyme adsorption to the cell surface were discussed.

Specificity of trypsin adsorption onto cellulose, glass, and quartz

Specificity of trypsin adsorption onto cellulose, glass, and quartz

The surface of the washed human erythrocyte as a polyanion.

The electrokinetic behaviour of normal erythrocytes is compared with that of trypsin-, N-bromosuccinimide-, and tosyl-treated erythrocytes. Reduction in the net negative charge with reduction in ionic strength of the suspending medium and also on treatment with N-bromosuccinimide and trypsin is discussed using a porous non-rigid polyanion as a model for the periphery of the cell membrane. It is deduced from the equivalent binding of chloride and thiocyanate ions and the absence of any effect on treatment of red cells with tosyl chloride, that normal, N-bromosuccinimide- and trypsin-treated cells are polyanionic in character. Reduction in erythrocyte charge on treatment with N-bromosuccinimide or trypsin is probably not due to the removal of phosphate groups from the interface, nor to physical adsorption of N-bromosuccinimide or trypsin. The charge reduction is probably produced by bond fission with possibly a net disappearance of carboxyl groups from the electrophoretic plane of shear either by loss from, or reorientation of, the membrane. The loss or reorientation of material associated with these carboxyl groups does not lead to any basic change in the character of the surface of the cell, nor to any obvious structural instability. The biconcave discoid form is maintained and there is no significant hemolysis of the erythrocytes even after contact with a solution of trypsin for 24 hours.

Activity of trypsin-lipid complexes at oil/water interfaces

Adsorption of trypsin at lipid-stabilized oil/water interfaces has been studied as a function of the nature of the interface. The adsorption of the enzyme, with corresponding changes in the activity expressed, is found to be related to the charge and nature of the lipid polar groups at the interface and to the charge and nature of the polar groups of the protein. Interactions between enzyme and lipid at the interface are paralleled in bulk with soluble lipid homologs. The loss in activity of the enzyme at the interfaces studied is thought to be due to a general structural unfolding of the molecule. Provided that the unfolding process has not gone too far, desorption of the surface enzyme can lead to partial recovery of activity. Some of the problems involved in the study of the effect of changes in the general structure of the protein on the enzymatic activity by the present methods are discussed. It is concluded that a determination of whether or not these partially “denatured” enzymes can work at lower efficiency necessitates careful investigation of the kinetic and thermodynamic properties of the adsorbed, partially unfolded enzyme.

Activity of trypsin-lipid complexes at oil/water interfaces

Adsorption of trypsin at lipid-stabilized oil/water interfaces has been studied as a function of the nature of the interface. The adsorption of the enzyme, with corresponding changes in the activity expressed, is found to be related to the charge and nature of the lipid polar groups at the interface and to the charge and nature of the polar groups of the protein. Interactions between enzyme and lipid at the interface are paralleled in bulk with soluble lipid homologs. The loss in activity of the enzyme at the interfaces studied is thought to be due to a general structural unfolding of the molecule. Provided that the unfolding process has not gone too far, desorption of the surface enzyme can lead to partial recovery of activity. Some of the problems involved in the study of the effect of changes in the general structure of the protein on the enzymatic activity by the present methods are discussed. It is concluded that a determination of whether or not these partially “denatured” enzymes can work at lower efficiency necessitates careful investigation of the kinetic and thermodynamic properties of the adsorbed, partially unfolded enzyme.

The Adsorption and Reactions of Enzymes and Proteins on Kaolinite. II

AbstractSome preliminary data illustrating the role of adsorption of enzymes and proteins to kaolinite in enzyme‐substrate reactions are presented. The adsorption of trypsin and chymotrypsin on kaolinite is nearly complete within 2–3 minutes. Over 40 mg. of chymotrypsin (MW 27,000) is adsorbed per gram of kaolinite whereas only 28 mg. of lysozyme (MW 15,000) is adsorbed. Much less chymotrypsin is adsorbed on kaolinite previously nearly covered by lysozyme. Heat‐inactivated lysozyme either in solution or adsorbed on kaolinite is subject to hydrolysis by chymotrypsin. The rate of hydrolysis is slower in the adsorbed state, however. Adsorbed trypsin is slowly hydrolyzed by free trypsin in a suspension of kaolinite. Thus substrates adsorbed on kaolinite are subject to hydrolysis by enzymes in solution. Lysozyme and trypsin eluted from kaolinite with ethylamine are active as enzymes. The possible significance of such findings for an understanding of the microbial utilization of macromolecules in the presence of clay minerals is discussed.