Solution structure of an amyloid-forming protein during photoinitiated hexamer-dodecamer transitions revealed through small-angle neutron scattering.

Shape-reconstruction analysis applied to small angle neutron scattering (SANS) data is used to determine the in vitro conformations of alpha-chymotrypsin oligomers that form as a result of partial unfolding with a photoresponsive surfactant. In the presence of the photoactive surfactant under visible light, the native oligomers (dimers or compact hexamers) rearrange into expanded corkscrew-like hexamers. Converting the surfactant to the photopassive form with UV light illumination causes the hexamers to laterally aggregate and intertwine into dodecamers with elongated, twisted conformations containing cross-sectional dimensions similar to amyloid protofilaments. Secondary-structure measurements with FT-IR indicate that this photoinduced hexamer-to-dodecamer association occurs through intermolecular beta sheets stabilized with hydrogen bonds, similar to amyloid formation. Traditional structural characterization techniques such as X-ray crystallography and NMR are not easily amenable to the study of these non-native protein conformations; however, SANS is ideally suited to the study of these associated intermediates, providing direct observation of the mechanism of oligomeric formation in an amyloid-forming protein. Combined with photoinitiated hexamer-to-dodecamer associations in the presence of the photoresponsive surfactant, this study could provide unique insight into the amyloidosis disease pathway, as well as novel disease treatment strategies.

A simple procedure for the photoregulation of chymotrypsin activity.

A convenient and rapid method for the photo-regulation of the proteolytic enzyme alpha-chymotrypsin is described. When alpha-chymotrypsin is coated with photolytic 1-(2-nitrophenyl)ethanol residues this not only markedly reduces the capability of the enzyme to digest both of the small substrates N-benzoyl-L-tyrosine ethyl ester and N-succinyl-L-phenylalanine p-nitroanilide, but also completely inhibits the enzyme's proteolytic activity. The inactivated alpha-chymotrypsin can then be reactivated under physiological conditions, when and where it is required, by exposure to UV-A light. These results further demonstrate that 1-(2-nitrophenyl)ethanol coated proteins can often be used as light sensitive biological switches as a simple alternative to site directed procedures.

Enhancing enzyme stability against TiO2-UV induced inactivation.

The use of enzymes in conjunction with inorganic photocatalysts requires stability against photooxidation. In this paper, we describe enhanced stabilization of a model enzyme, chymotrypsin, to photooxidation driven by titanium dioxide exposed to ultraviolet light (TiO(2)-UV). Stabilization is achieved conjugating the enzyme with an oligomeric adduct of UV-absorbing (2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate) (HBMA) and free radical-absorbing 2-methacryloyloxyethyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox-HEMA). Juxtaposition of the antioxidant Trolox with the UV absorber HBMA within a single chain reduced the rate of deactivation of the former by TiO(2)-UV. This enables modified enzyme, which is adsorbed on TiO(2), to absorb both UV-light and free radicals and locally reduce the rate of photooxidation. Interestingly, Trolox was more readily deactivated by TiO(2)-UV when it was conjugated separately to chymotrypsin that had been pre-modified with HBMA moieties.

Rational protein modification leading to resistance of enzymes to TiO2-UV irradiation-induced inactivation.

Photoexcited TiO2 degrades biomolecules such as nucleic acids, cell membrane proteins, and enzymes. Stabilization of enzyme activity against the deactivation caused by the combination of TiO2-UV is essential if we are to develop novel hybrid materials exhibiting photocatalytic and biocatalytic activities useful for decontamination applications. In this paper we describe the stabilization of a model enzyme, chymotrypsin, against TiO2-UV-induced deactivation by conjugating the enzyme with UV-absorbing, carboxyl-terminated oligo[2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate] [oligo(HBMA)-COOH]. Chymotrypsin was completely deactivated within 3 h, whereas the chymotrypsin-oligo(HBMA) conjugate retained > 50% activity even after 5 h of exposure to TiO2-UV (lambdamax 365 nm). The degree of enzyme stabilization induced by the conjugated UV absorber was 2-fold higher than that from the equivalent number of conjugated PEG chains. Spectroscopic characterizations revealed that chymotrypsin-oligo(HBMA) absorbs UV light and initially resists photoexcitation of TiO2. Modified chymotrypsin also exhibited resistance to changes in the secondary structure during the deactivation. This method of stabilizing enzymes against photodegradation could be also useful in photolithographic enzyme immobilizations for sensors and arrays or for stabilization of any UV-sensitive protein.

A biochemical logic gate using an enzyme and its inhibitor. Part II: The logic gate.

Enzyme-Based Logic Gates (ENLOGs) are key components in bio-molecular systems for information processing. This report and the previous one in this series address the characterization of two bio-molecular switching elements, namely the alpha-chymotrypsin (alphaCT) derivative p-phenylazobenzoyl-alpha-chymotrypsin (PABalphaCT) and its inhibitor (proflavine), as well as their assembly into a logic gate. The experimental output of the proposed system is expressed in terms of enzymic activity and this was translated into logic output (i.e. "1" or "0") relative to a predetermined threshold value. We have found that an univalent link exists between the dominant isomers of PABalphaCT (cis or trans), the dominant form of either acridine (proflavine) or acridan and the logic output of the system. Thus, of all possible combinations, only the trans-PABalphaCT and the acridan lead to an enzymic activity that can be defined as logic output "1". The system operates under the rules of Boolean algebra and performs as an "AND" logic gate.

Modification of alpha-chymotrypsin using a water-soluble photo-Fenton reagent.

Modification of an enzyme, alpha-chymotrypsin, was examined by using a water-soluble photo-Fenton reagent. By photoirradiation of the enzyme with the reagent, which can occupy a binding site of the enzyme, a tryptophan residue in the vicinity of the active site was oxidized to N-formylkynurenine. Concurrently, the catalytic properties of the enzyme were largely changed: the Km was increased and the kcat was decreased. The decrease in kcat for a specific amide substrate was the most significant among the esters and amides examined. The water-soluble photo-Fenton reagent would be useful to chemically modify relatively limited regions in biomolecules.

Photoregulation of alpha-chymotrypsin activity in organic media: effects of bioimprinting.

alpha-Chymotrypsin exhibits photoswitchable activities in an organic solvent after covalent modification of the protein backbone with thiophenefulgide active ester (2). The thiophenefulgide-modified alpha-chymotrypsin exhibits reversible photoisomerizable properties between states (3)-E and (3)-C. The modified alpha-chymotrypsin, where nine lysine residues are substituted by thiophenefulgide units, retains 60% of the activity of the native enzyme. The activities of thiophenefulgide-modified alpha-chymotrypsin toward esterification of N-acetyl-L-phenylalanine (4) by ethanol in cyclohexane are controlled by the configuration of the attached photoisomerizable component and by prior bioimprinting of the protein backbone with the reaction substrate (4). The esterification of (4) in cyclohexane using bioimprinted (3)-C is two-fold faster than in the presence of (3)-E. In the presence of a nonbioimprinted enzyme, esterification of (4) by (3)-C is five-fold faster than with (3)-E. The activity of bioimprinted (3)-E toward esterification of (4) is 4.5-fold higher than that of nonbioimprinted (3)-E. Switchable cyclic esterification of (4) is accomplished by sequential photoisomerization of the thiophenefulgide-modified alpha-chymotrypsin between states (3)-C and (3)-E.

Study of the photochemical immobilization of alkaline proteinase and chymotrypsin on the solid phase of O-hydroxyethylcellulose.

Stability of the photochemically immobilized alkaline proteinase (E.C. 3.4.21.14) and chymotrypsin (E.C. 3.4.21.1.) onto the gel of O-hydroxyethylcellulose has been studied. For the purpose of immobilization the photochemical generation of nitrene radicals caused by the photolysis of an azido group of bifunctional 4,4'-bis-azidostilbene-2,2'-disodium-sulphate and the newly synthetized O-(3-azidophthaloyl)-O-hydroxyethylcellulose have been employed. The immobilized alkaline proteinase demonstrated a decreased ability of denaturation and an increased laboratory stability.

[Radiation inactivation of alpha-chymotrypsin in aqueous solutions. The nature of the dose-response relationship]. / Radiatsionnaia inaktivatsiia al'fa-khimotripsina v vodnykh rastvorakh. Kharakter zavisimosti éffekt--doza.

A study was made of possible reasons for deviations of the dose--response curves from the exponential function at low (less than or equal to 10(-6) M) and high (greater than or equal to 10(-4) M) initial concentrations of the enzyme. Factors influencing the degree of the deviation and type of the dependence of the radiation and chemical yield on the initial concentration of the enzyme are discussed. The data obtained are compared with those reported in the literature.

[Radiation inactivation of alpha-chymotrypsin in aqueous solutions. Effect of irradiation conditions]. / Radiatsionnaia inaktivatsiia alpha-khimotripsina v vodnykh rastvorakh.Vliianie uslovii oblucheniia.

A comparative study was made of inactivation by gamma- and beta-radiation of alpha-chymotrypsin within a wide range of its initial concentrations (from 10(-4) to 10(-7) M). The regularities of gamma- and beta-inactivation are the same, and distinctions, if any, are due to a greater radiation effect of beta-rays on dilute enzyme solutions (less than or equal to 5 X 10(-6) M). The inactivation of alpha-chymotrypsin by radiation proceeds either via primary molecule unfolding followed by degradation of the most accessible and radiosensitive amino acid residues (pH 7.8) or, to a greater extent, via direct disruption of amino acid residues which can probably be random (pH 3.0). Calcium ions stabilize, on the whole, the enzyme molecule upon irradiation.

Behavior of enzyme activity in immobilized proteases.

Proteases such as trypsin, alpha-chymotrypsin, papain, and thermolysin were immobilized by radiation polymerization of various monomers at low temperatures, and behavior of enzyme activity in immobilized proteases was studied. The enzyme activity in immobilized proteases appeared to be different by the kind of proteases; the order of the magnitude of the enzyme activity was papain greater than trypsin greater than thermolysin greater than alpha-chymotrypsin. This difference of the enzyme activity was explained by the change of the molecular conformation in enzyme reaction.

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin.

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.

Light-induced enzyme system leading to pigmentation.

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.

An improved method of light-induced pigmentation.

An improved procedure was developed whereby a primary light signal can be intensified and made visible by activation of a pre-tyrosinase (pre-phenoloxidase) enzyme [isolated from silkworm (Bombyx mori)] by alpha-chymotrypsin; this activation results from the light-activated conversion of the inactive cis-cinnamoyl-alpha-chymotrypsin.