Kinetics Of The Interaction Of Bovine Pancreatic Trypsin Inhibitor (Kunitz) With α-Chymotrypsin

Quast, Urich; Engel, Juergen; Heumann, Hermann; Krause, Guenther; Steffen, Erna

doi:10.1021/bi00709a600

Cited by 73 publications

(59 citation statements)

References 23 publications

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“…At a positive enthalpy change the process is entropy driven, which is probably explained by the predominance of hydrophobic interaction [14]. It was also observed in another system [15] that the enthalpy change becomes more negative when specific interactions, such as hydrogen bonds, develop. X-ray crystallographic studies on the variable fragment of the Bence-Jones protein Au prove the presence of dimers in the crystalline state [16].…”

Section: Discussionmentioning

confidence: 82%

Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au

Maéda¹,

Engel²,

Schramm³

1976

European Journal of Biochemistry

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The dimerization of the variable fragment of the Bence-Jones protein Au was examined in phosphate buffers at pH 6.8 -6.9 and ionic strength of 0.1 M or 0.2 M at 20 "C. The dimerization constant was about 1 x 10' M-'. The reaction enthalpy was positive and the process was entropy driven. The association and dissociation rate constants were 9 x lo6 M-' s-' and 1.5 x lo2 s-l respectively. Temperature-jump experiments exhibited the presence of two isomers of the dimer, which are present at equilibrium in a ratio of about 1 : 1. Isomerization occurred with a half-life of about 0.1 s.Recent X-ray crystallographic studies have shown that immunoglobulin molecules consist of several domains and implied the presence of non-covalent interactions between the homologous domains on different chains [l, 21. These interactions can be studied with isolated fragments. The association between heavy and light chains [3,4] and the dimerization of light chains [5,6] have been studied. The study of the dimerization of the variable fragment will provide information about the interactions between constant domains as well as those between variable domains, when combined with the studies on the whole light chains [5,6]. In the present study both equilibrium and kinetic aspects of dimerization of the variable part of a Bence-Jones protein (Au) were examined. Such data are of interest for an understanding of the interaction between the different domains in a Bence-Jones protein and possibly in immunoglobulin G. It was also hoped that the study may contribute to the important question [1,2] of whether distinct conformational isomers exist for the domains. MATERIALS AND METHODSThe variable part of the Bence-Jones protein Au was prepared as reported [7]. Both the fraction 111 obtained by Sephadex chromatography (G-75, eluent : 0.5 M acetic acid) and the fraction obtained by trypsin digestion were used. Identical results were obtained with both materials. The samples were additionally purified by DEAE-cellulose chromatography (gradient: 0.05-0.4 M Tris-HCI pH = 8.0). These samples showed only one band in dodecylsulfate polyacrylamide electrophoresis. Other chemicals were from Merck and used without further purification.A phosphate buffer solution 0.025 M KH2P04+ 0.025 M Na2HPO4 (pH = 6.9, I = 0.1 M) was used throughout the equilibrium measurements unless otherwise stated. In the kinetic measurements buffer solution of double concentration was used (pH = 6.8, 1 = 0.2 M). The total protein concentration is defined by cp = 2 CD + cM, where L'U (cM) are molar concentrations of dimers (monomers). The concentration of the protein was calculated from the absorbance at 278 nm using a molar absorption coefficient per monomer of 2 . 0~ lo4 M-' cm-'. This value was determined on a weight basis for the solutions of a concentration range of 0.01 -0.02 mM. The effect of dimerization reaction was corrected if necessary. The sedimentation equilibrium measurements were made with a Beckman Spinco Model E analytical ultracentrifuge equipped with a scanner. Rotor ...

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Section: Discussionmentioning

confidence: 82%

Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au

Maéda¹,

Engel²,

Schramm³

1976

European Journal of Biochemistry

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“…It was characterized as described before [12,14]. The molar absorption coefficient was E~~~ = 5.4 x lo3 M -cm-'.…”

Section: Methodsmentioning

confidence: 99%

“…The data were stored with a transient recorder (Datalab DL 905) which was connected on line to a PDP 11/40 computer. The proflavine displacement method [12] was used to follow the association of c(-chymotrypsin with inhibitor. The absorbance change due to the displacement of proflavine by the inhibitor is proportional to the change of free enzyme concentration [12] and was monitored at 465nm.…”

Section: Methodsmentioning

confidence: 99%

“…The proflavine displacement method [12] was used to follow the association of c(-chymotrypsin with inhibitor. The absorbance change due to the displacement of proflavine by the inhibitor is proportional to the change of free enzyme concentration [12] and was monitored at 465nm. All association rate constants as well as equilibrium constants which were determined by this method were corrected for the fraction of enzyme occupied by proflavine [12].…”

Section: Methodsmentioning

confidence: 99%

“…In the reaction with virgin inhibitor, Cis formed via a Michaelis complex L, which is in fast pre-equilibrium with inhibitor and enzyme with an equilibrium constant KL = 5 x M [12]. From I* formation of C is about 2 x lo4 times slower than from I [8].…”

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confidence: 99%

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Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Quast¹,

Engel²,

Steffen³

et al. 1978

European Journal of Biochemistry

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Modified trypsin kallikrein inhibitor (I*), with the reactive‐site peptide bond Lys‐15–Ala‐16 split, reacts with α‐chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C: E + I*⇌ X → C. Formation of X constitutes a fast pre‐equilibrium (equilibrium constant Kx= 7 × 10−5 M. association rate constant kx= 4 × 103 M−1 s−1) to the slow reaction X → C (rate constant kc= 2 × 10−3 s−1), all values at pH 7.5. No intermediate X is observed when α‐chymotrypsin reacts with I*‐OMe in which the carboxyl group of Lys‐15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*‐OMe indicate that formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with β‐trypsin and I* or I*‐OMe.

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Multiple intermediates in the reaction of bovine β‐trypsin with bovine pancreatic trypsin inhibitor (kunitz)

et al. 1983

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SynopsisThe kinetics of the formation of the complex between bovine P-trypsin and the bovine basic pancreatic trypsin inhibitor (BPTI) was investigated using three different signals: the displacement of proflavine, the optical density changes in the UV region, and the loss of the enzymatic activity. For the three different signals, with inhibitor in excess over bovine, the time course of the reaction corresponds to a pseudo-first-order process. The concentration dependence of the rate is second order at low BPTI concentrations and tends to first order a t high inhibitor concentrations. This hehavior may he explained by relatively rapid preequilihria followed by limiting first-order processes according toThe values of K,, k+,, and k(,,,, ( = k+,/K,) have been determined for the different reactions at three pH values: 6.80,4.80, and 3.50. The kinetic parameters differ widely for the processes reflected by the various signals; the difference increases upon lowering pH. The results indicate that the formation of the bovine P-trypsin-BPTI complex is not an all-or-nothing process, but involves several intermediates corresponding to discrete reaction steps, which are differently affected by ionization processes.

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Kinetics Of The Interaction Of Bovine Pancreatic Trypsin Inhibitor (Kunitz) With α-Chymotrypsin

Cited by 73 publications

References 23 publications

Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au

Kinetics of Dimerization of the Variable Fragment of the Bence‐Jones Protein Au

Kinetics of the Interaction of α‐Chymotrypsin with Trypsin Kallikrein Inhibitor (Kunitz) in Which the Reactive‐Site Peptide Bond Lys‐15–Ala‐16 is Split

Multiple intermediates in the reaction of bovine β‐trypsin with bovine pancreatic trypsin inhibitor (kunitz)

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