Modified proteins, preparation

Limited digestion of globular soy proteins with rennin affords a modified protein preparation which retains a high molecular weight (47). Whipping quality, measured by foam volume and stability, was superior in comparison with native proteins. The limited rennin proteolysis of soy was identified as a key factor in functionality, since this modification conferred improved solubility. 

Limited digestion of the globular soy proteins (Harosoy variety) with the enzyme rennin affords a modified protein preparation which retains a high molecular weight. The enzyme-modified protein is precipitated and washed with alcohol and subsequently heat-modified. A flavorless product results which is easily dispersed in water and shows excellent functional characteristics. 

We used an anti-DNA antibody as an exploratory model system. The antibody was monoelonal from mouse sourees and its subelass was IgM. Mouse IgG (MW 1.5 x 10 Da) and IgM (MW 9 X 10 Da) antibodies from normal plasma, and bovine serum albumin were used for the eontrol measurements. To prevent the nonspeeilie adsorption of proteins to the uneovered, bare Au site in the modified eleetrode surfaee, the DNA-modified eleetrode prepared by the standard proeedure was further treated with aqueous 2-mercaptoethanol solution and was used for the measurements. 

Preparations of PEG-modified proteins. A. SC-PEG (1 g, 0.2 mmol) was added to a stirred solution of Bovine Serum Albumin (BSA) (100 mg, 1.5 x 10 6 mol) in 0.1 M sodium phosphate, pH 7.8 (60 mL). Sodium hydroxide (0.5 N) was used to maintain pH 7.8 for 30 min. The excess of free PEG was removed by diafiltration using 50 mM phosphate buffered saline. Approximately 30 amino groups of the native protein were modified as determined by trinitrobenzenesulfonate (TNBS) assay (28). The same degree of modification was obtained when the experiment was repeated under identical conditions using SS-PEG instead of SC-PEG. 

To release the pyridine-2-thione leaving group and form the free sulfhydryl, add DTT at a concentration of 0.5 mg DTT per mg of modified protein. A stock solution of DTT may be prepared to make it easier to add it to a small amount of protein solution. In this case, dissolve 20mg of DTT per ml of 0.1M sodium acetate, 0.1M NaCl, pH 4.5. Add 25 pi of this solution per mg of modified protein. Release of pyridine-2-thione can be followed by its characteristic absorbance at 343 nm (s = 8.08 X 103M 1cm 1). 

Add an aliquot of the hydrazine-modified protein solution to the p-nitrobenzaldehyde solution and incubate at 37°C for 1 hour or at room temperature for 2 hours. To assure accuracy, determine the linear response range of the test by adding a series of different concentrations of the hydrazine-modified protein solution to the p-nitrobenzaldehyde buffer. This is done by preparing a set of serial dilutions of the protein solution and... 

Prepare a 1M solution of hydroxylamine in coupling buffer sufficient to again treat the slide. The pH of the coupling buffer should be adjusted to pH 10 after dissolving the hydroxylamine into it. Expose the slide to the hydroxylamine solution in the same manner as the crosslinker treatment. The hydroxylamine will react with the methyl ester groups on the salicylic acids and form hydroxamate functionalities suitable for conjugation with the P(D)BA-modified protein from above. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Chemical perturbations can be a useful approach to this problem. In laccase, selective replacement of the type 1 coppo- with mercury has allowed the type 1 site in laccase to be characterized stmcturally, even in the presence of three other copper atoms (27). Likewise, Simolo et al. have studied independently the a and 3 subunits in hemoglobin by preparation of the (a-M)2(P-M )2 derivaties, where M and M are different metals (28). An alternative to replacement is metal removal, for example Cu in cytochrome-c oxidase (29) or the T3 Ci in laccase (30). The concern with all of these approaches is to establish that the modified protein has the same metal-site structures as the native protein. 

Preparation of Protease-Treated Proteins. The proteolysis of soy isolate was carried out by introducing 6 mL pronase E (mg/mL) to a well dispersed mixture of 12 g Mira Pro 111 in 760 mL water. Different levels of proteolysis were achieved by reaction at 50 °C for various periods of time. The reaction was stopped by heating at 100 C for 3 min, and the modified protein was then recovered by lyophilization. 

Willner et al. also prepared fulgimide-modified (through lysine nitrogen) a-chy-motrypsin 30, with nine fulgimide molecules in one protein. 311 The modified protein was active towards esterification of N-acetylphenylalanine in cyclohexane. Together with the bioimprinting technique of the substrate, the rate of esterification could be accelerated by irradiation with UV light. 

In Ref. [279] the technique of protein modification was used to study the dependence of the rate of photoinduced electron tunneling on the distance between TZnP and Ru(III) sites in modified myoglobins. The modified proteins were prepared by substitution of zinc mesoporphyrin IX diacid for the heme in four various pentaammineruthenium (III) derivatives of sperm whale myoglobin (NH3)5Ru(His-48)Mb, (NH3)5Ru(His-12)Mb, (NH3)5Ru(His-116)Mb and (NH3)5Ru(His-81)Mb. Metal-to-metal distance between ZnP and (NH3)5Ru(His) ranges in this seria from 16.1-18.8 A for His-48 to 27.8-30,5 for His-12. The rate constant of electron tunneling decreases in this series in accordance with Eq. (1) with ve = 7.8 x 10s s 1 and ae = 2.2 A at T = 298 K. 

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