Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering

G. Jason Wei, Victor A. Bloomfield, Robert M. Resnick, Gary L. Nelsestuen

Research output: Contribution to journalArticle

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Abstract

We have investigated the kinetics and mechanism of prothrombin-membrane vesicle interaction by using stopped-flow light scattering. Under conditions of approximately physiological protein concentration (≤3 μM prothrombin), prothrombin interaction with the vesicles was modeled according to a simple bimolecular process with noninteracting prothrombin binding sites on the vesicle. The association rate constant (per protein binding site) for interaction of prothrombin with vesicles containing 20% phosphatidylserine-80% phosphatidylcholine at 10 °C, in buffer containing 3 mM calcium, is (1 ± 0.1) × 107 M-1·s-1. This corresponds to a 10% collision efficiency. The reverse process is a first-order dissociation with a rate constant of 3 ± 1 s-1. Off-rate experiments conducted by sample dilution were consistent with these values. With varying membrane compositions the association process was found to be somewhat cooperative with respect to phosphatidylserine, but dissociation was unaffected by phosphatidylserine density. The activation energy for prothrombin-membrane association varied with the amount of acidic phospholipid in the membrane. Membranes of 10% phosphatidylserine gave an activation energy of about 9 kcal/mol while those of 40% phosphatidylserine gave a value of 4 kcal/mol. For these same membranes the collision efficiency was estimated to be 3 and 20%, respectively. This trend in the activation energy suggests that as the acidic phospholipid content increases, the association energy of activation becomes characteristic of a diffusion-controlled reaction. Dissociation rate constants were obtained by mixing prothrombin-membrane complexes with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). It was found that a necessary population of calcium ions exchanged rapidly from the prothrombin-membrane complex but that this population could be replaced by magnesium or manganese. A second population of essential ions, specific for calcium, exchanged slowly and, under certain conditions, appeared to be released at the rate of prothrombin-membrane dissociation. Under conditions of high free-protein concentrations the association process became complex and had lower rate constants. The anomalous binding characteristics were observed under conditions that are not likely to be physiologically important.

Original languageEnglish (US)
Pages (from-to)1949-1959
Number of pages11
JournalBiochemistry
Volume21
Issue number8
StatePublished - 1982

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Prothrombin
Light scattering
Membranes
Light
Phosphatidylserines
Kinetics
Association reactions
Rate constants
Activation energy
Calcium
Phospholipids
Binding Sites
Ions
Population
Ethylene Glycol
Egtazic Acid
Manganese
Phosphatidylcholines
Protein Binding
Ether

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Wei, G. J., Bloomfield, V. A., Resnick, R. M., & Nelsestuen, G. L. (1982). Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering. Biochemistry, 21(8), 1949-1959.

Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering. / Wei, G. Jason; Bloomfield, Victor A.; Resnick, Robert M.; Nelsestuen, Gary L.

In: Biochemistry, Vol. 21, No. 8, 1982, p. 1949-1959.

Research output: Contribution to journalArticle

Wei, GJ, Bloomfield, VA, Resnick, RM & Nelsestuen, GL 1982, 'Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering', Biochemistry, vol. 21, no. 8, pp. 1949-1959.
Wei GJ, Bloomfield VA, Resnick RM, Nelsestuen GL. Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering. Biochemistry. 1982;21(8):1949-1959.
Wei, G. Jason ; Bloomfield, Victor A. ; Resnick, Robert M. ; Nelsestuen, Gary L. / Kinetic and mechanistic analysis of prothrombin-membrane binding by stopped-flow light scattering. In: Biochemistry. 1982 ; Vol. 21, No. 8. pp. 1949-1959.
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abstract = "We have investigated the kinetics and mechanism of prothrombin-membrane vesicle interaction by using stopped-flow light scattering. Under conditions of approximately physiological protein concentration (≤3 μM prothrombin), prothrombin interaction with the vesicles was modeled according to a simple bimolecular process with noninteracting prothrombin binding sites on the vesicle. The association rate constant (per protein binding site) for interaction of prothrombin with vesicles containing 20{\%} phosphatidylserine-80{\%} phosphatidylcholine at 10 °C, in buffer containing 3 mM calcium, is (1 ± 0.1) × 107 M-1·s-1. This corresponds to a 10{\%} collision efficiency. The reverse process is a first-order dissociation with a rate constant of 3 ± 1 s-1. Off-rate experiments conducted by sample dilution were consistent with these values. With varying membrane compositions the association process was found to be somewhat cooperative with respect to phosphatidylserine, but dissociation was unaffected by phosphatidylserine density. The activation energy for prothrombin-membrane association varied with the amount of acidic phospholipid in the membrane. Membranes of 10{\%} phosphatidylserine gave an activation energy of about 9 kcal/mol while those of 40{\%} phosphatidylserine gave a value of 4 kcal/mol. For these same membranes the collision efficiency was estimated to be 3 and 20{\%}, respectively. This trend in the activation energy suggests that as the acidic phospholipid content increases, the association energy of activation becomes characteristic of a diffusion-controlled reaction. Dissociation rate constants were obtained by mixing prothrombin-membrane complexes with ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). It was found that a necessary population of calcium ions exchanged rapidly from the prothrombin-membrane complex but that this population could be replaced by magnesium or manganese. A second population of essential ions, specific for calcium, exchanged slowly and, under certain conditions, appeared to be released at the rate of prothrombin-membrane dissociation. Under conditions of high free-protein concentrations the association process became complex and had lower rate constants. The anomalous binding characteristics were observed under conditions that are not likely to be physiologically important.",
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