A simple preparation of β-trypsin based on a calorimetric study of the thermal stabilities of α- and β-trypsin

Beardslee, R A; Zahnley, James C.

doi:10.1016/0003-9861(73)90575-4

Cited by 18 publications

(1 citation statement)

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“…The reason could be the inhomogeneity of commercial preparations of trypsin. They contain at least two active forms (a-and /?-trypsin) which are not structurally identical [41] and hence differ in stability [42]. The amination of tyrosine residues of trypsin slows down the inactivation of both fractions (Fig.…”

Section: Nitration and Amination Of Tyrosine Residues In Trypsinmentioning

confidence: 99%

Protein stabilization via hydrophilization

Mozhaev

Šikšnis

Melik‐Nubarov

et al. 1988

European Journal of Biochemistry

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This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach.1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one.2. a-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of cr-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65 -98 "C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.Applications of enzymatic catalysis in biotechnology, fine organic synthesis, analysis, medicine, and other areas are often hindered because many enzymes, if isolated from their natural environment in vivo, become unstable and rapidly inactivate (denature); for review, see [l, 21. For these reasons the problem of enzyme stabilization has received considerable attention in recent years [3 -81.The analysis of the structure-stability relationships in the protein leads us to the conclusion [S] that the structure of proteins is not optimal with regard to their stability. There are still some reverses for stabilization which are used by nature for the production of extremely stable proteins, such as, for example, enzymes from thermophilic microorganisms ; for review, see [S, 91. Reserves of such a kind may be useful for artificial stabilization of enzymes. We shall discuss here, from this viewpoint, how to improve the protein structure in order to make enzymes more stable.According to X-ray crystallographic data, about one half of the surface area of proteins is occupied by nonpolar amino Corre2pondence to K. Martinek, Ustav OrganickC Chemie a Biochemie, Ceskoslovenska Akademie Vtid, Flemingovo nimEsti 2, CS-166-10 Praha, CzechoslovakiaThe authors wish to dedicate this paper to the memory of their teacher and friend. Professor...

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Section: Nitration and Amination Of Tyrosine Residues In Trypsinmentioning

confidence: 99%