Kinetic properties of subtilisin type Carlsberg in the crystalline state

Self Cite

A method is presented for measurement of hydrogen-tritium exchange in insoluble protein samples using a specially constructed closed chamber type filtration device. The protein sample is initially exhaustively tritiated at high pH, and back-exchange is initiated in the filtration unit by removal of tritiated solvent from the sample by rinsing with buffer of desired pH. The back-exchange is followed by sequential samplings of the tritium liberated to the solvent and it is completed by increasing pH. Experiments with monoclinic hen egg white lysozyme crystals, insolubilized by cross-linking with glutaraldehyde, could be followed as early as 20 seconds from initiation of back-exchange and exchange curves were highly reproducible. Exchange curves were identical with those of the dissolved enzyme at pH 3 and only small differences were detected between the two states at pH 6.8, supporting the idea of negligible change of protein motility upon crystallization and cross-linking. Diffusional equilibration processes are discussed.

Section: Initial Rinsing Proceduressupporting

confidence: 87%

“…The polymer was prepared according to MONSAN (12) and this substance has been suggested to be homologous with the actual crosslinking agent (12,21). At pH 4.7 no measurable exchange was detected, while on complete back-exchange in 0.…”

Section: Liberation Of Residual Tritium At Completion Of Experimentsmentioning

confidence: 99%

A simple hydrogen exchange method for cross-linked protein crystals

Tüchsen

Ottesen

1979

Self Cite

“…The crystals from sodium sulphate were cross-linked by treatment with 1% glutaraldehyde at pH 5.5 as previously described (15). Before use in the ultracentrifuge the crystals of the stock suspensions were washed in small centrifuge tubes with solutions of CsCI of lower density than the solutions used to form the gradient.…”

Section: Methodsmentioning

confidence: 99%

“…The protein content of the crystals and the extent of lysyl residue modification, which is supposed to reflect the degree of cross-linking, were determined by amino acid analysis (15). Samples of crystals: containing about 1 mg of protein were hydrolyzed with 1 ml 6M-HCI in evacuated, sealed tubes for 24 hours at 110~ and analyzed in a Durrum model 500 amino acid analyzer.…”

Section: Methodsmentioning

confidence: 99%

Buoyant densities of subtilisin Carlsberg in dissolved and crystalline states

Pedersen

Tüchsen

Svendsen

et al. 1978

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The buoyant densities of subtilisin Carlsberg in dissolved and crystalline states, including glutaraldehyde crosslinked crystals, have been determined by equilibrium centrifugation at neutral pH in the analytical ultracentrifuge in concentrated solutions of CsCI. Due to the negligible diffusion of suspended crystals they form a sharp band at the position in the gradient corresponding to their density. Different crystal preparations may be separated in a single gradient when their densities differ by more than 0.005 g'ml -~ and the results are reproducible to -+0.001 g-m1-1. Glutaraldehyde stabilized crystals were found to have higher buoyant densities than crystals of unmodified subtilisin. The increase in density due to the glutaraldehyde cross-linking can be explained if a polymer composed of approximately 5 glutaraldehyde residues is bound per pair of blocked lysinr residues. Unmodified subtilisin Carlsberg shows identical densities in the crystalline and soluble states in CsCI solutions. The observation is consistent with identical molar volumes of subtilisin Carlsberg in solution and crystals and no charged groups being involved in intermolecular binding in the crystals.

“…Recent reports concerning the properties of crystalline enzymes have compared the kinetic parameters of cross-linked insoluble crystals with those of the corresponding soluble enzymes (1 I, 15,18). In some cases, crystallization decreases kca t with small changes of Kin; in others, only small changes in both kca t and K m have been noted.…”

Section: Introductionmentioning

confidence: 99%

Properties of lactate dehydrogenase crystals reacted with diimidoesters

Bayne

Ottesen

1977

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Pig heart lactate dehydrogenase crystals were reacted under mild conditions in saturated sodium sulphate solution with a homologous series of diimidoesters varying in chain length from five to twelve carbon atoms. With diimidoesters of chain length of eight or nine carbon atoms the crystals were stabilized, with less than seven carbon atoms, the reacted crystals dissolved, whereas with diimidoesters of ten to twelve carbon atoms, mixtures of amorphous insoluble material and crystals resulted. The physical appearance of the enzyme was correlated with both its specific activity and degree of lysine modification. The MICHAELIS parameters were determined for crystals reacted with hexane-and octanediimidic acid dimethylesters and the values compared with those for unmodified lactate dehydrogenase. The K~ values for reduced coenzyme were increased in the cross-linked enzyme while the Km values for pyruvate remained virtually unchanged. Vm~ values were only affected to a minor extent for these two derivatives. The correlation between the ,static~ mechanism proposed by X-ray analysis and the reaction mechanism in dilute solution is discussed.