Anne D'Albis

Effet du pH sur la fixation d'inhibiteurs compétitifs synthétiques par la trypsine☆

Abstract Conformational changes in trypsin (EC 3.4.4.4) induced by the binding of competitive inhibitors (benzylamine, butylamine, N-α- benzoyl- l -arginine ) and, in some favourable cases, of substrates ( N-α- benzoyl- l -arginine ethyl ester, N-α- benzoyl- l -arginine amide) have been studied at 10°, in the pH range from 1.5 to 12.5. The formation of the trypsin-inhibitor (or trypsin-substrate) complex produces changes in the enzymes rotatory power; this modification is small at neutral pH but substantial in acid and alkaline pH ranges where trypsin is reversibly denatured. It seems that a more compact structure of the protein results from the formation of the complex. It has been shown by spectrophotometric measurements in alkaline medium that this conformational change is accompanied by the burying of one tyrosine residue which ionizes normally in the free enzyme. The formation of the enzyme-substrate complex coincides with a proton release at pH values lower than 6. Results obtained both by proton titration at pH 4.1 and by polarimetric measurement of the affinity of benzylamine for trypsin at pH 3.5 and 2.5 indicate the existence of an interaction between the positive charge of the inhibitor and the negative charge of a carboxyl group of the enzyme having a pK of 4.7. The role of the substrate in maintaining the structural and catalytic properties of trypsin is discussed.

Disuccinimidyl esters as bifunctional crosslinking reagents for proteins: Assays with myosin

Bifunctional crosslinking reagents have been used in studies of the spatial arrangement of muscle contractile proteins, such as myosin and actin, either in their soluble forms [ 1,2], or in synthetic filaments [3-51, or even in myofibrils [3]. The most commonly used reagents are the bisimidates, which are very reactive, but also quite unstable in aqueous solution; incomplete substitution and unexpected side reactions furthermore occur if the crosslinking reaction is at pH -8 [6,7]. A disuccinimidyl ester, the dithiobis (succinimidyl propionate), DSP, that does not have these drawbacks and contains, moreover, an easily cleavable disulfide bond, has been described [8]; unlike the bis-imidates, this reagent allowed the crosslinking of the two heads of a myosin molecule [ 21. Owing to the high chemical reactivity of DSP and its stability in water, we thought it interesting to synthesize a series of disuccinimidyl esters of various chain lengths (table 1). These include non-cleavable reagents (compounds I-IV), and also reagents with either a uic-glycol (compounds V, VI) or an ethylenic bond (compound VII); the crosslinks formed by these last compounds can in principle be cleaved,

Structural relationship of myosin isoenzymes: Proteolytic digestion patterns of heavy chain components from fast muscles, and comparison with other muscle types

Muscle myoslns are made up in all known cases of one pair of heavy chains and two pairs of light chains, the so-called alkali and regulatory subunits. Muscle type specificity of myosin has been ascribed to differences in the light and also the heavy subunits. indeed, the heavy chains of myosins from slow, fast, and cardiac muscles have been reported to differ immunologically [ 11, and the last two also chemically 12 3; these slow, fast and cardiac myosins are known to display different ATPase activities. Electrophore~s of myosin in its native state has demonstrated that in each of these muscles it is present in several isoenzymic forms [3,4]. In the case for instance of the fast-twitch muscles, such as chicken pectoralis and posterior latissimus dorsi or rabbit skeletal muscle, three myosin populations are found, which differ in their contents of the two alkali light chains, the subunits A, and Al. They contain, respectively, two Al, two AZ, and one of each light chain per molecule of myosin, and correspond therefore to Al and Aa homodimers and the AZ AZ heterodimer [4,5]. To determine whether the differences between these myosin components are confined to their alkali light chains, or extend also to the heavy chains, we have carried out a comparative study of the proteolytic digestion patterns of the latter from all the three fast muscle isoenzymes. After electrophoretic separation in non-dissociating conditions, the heavy chains of each resolved isoenzyme were separated from the light chains and recovered by way of a second electrophoresis

Binding of competitive inhibitors to the different pH-dependent forms of trypsin☆

Abstract Some of the rotatory, spectral, ionic and catalytic properties of trypsin (EC 3.4.4.4) have been studied, in the pH range from 1.5 to 12.5, in the presence and absence of synthetic competitive inhibitors. In the alkaline and acidic pH ranges where trypsin is reversibly denatured, the formation of the trypsin-inhibitor complex protects the enzyme against transconformation; at neutral pH, where trypsin is in the native form, the complex formation sometimes induces a change in the enzyme structure. In the alkaline pH range (a) the rotatory dispersion curve of the complex is pH independent; (b) the formation of the complex induces a decrease of the enzyme absorbance; (c) the formation of the complex produces a proton uptake by the enzyme; (d) the affinity of inhibitors decreases when the pH increases. All these results can be related to the ionization of an enzyme group having an apparent pK of about 10. In the acidic pH range (a) the rotatory power of the complex is pH independent, and (b) the complex formation produces a proton release by the enzyme. The changes in the ionic and rotatory properties of trypsin as a function of inhibitor concentration have allowed the evaluation of the dissociation constant of the benzamidine-trypsin complex between pH 4.5 and 1.5. A scheme for the interaction between the inhibitor and the different pH dependent forms of trypsin is proposed: it accounts for the pH dependence of the complex dissociation constant and also for the dependence of the number of protons released by the enzyme. The lowering of the inhibitor affinity in the acidic pH range is due to the protonation of two enzyme groups, one having an apparent pK of 3.7, the other a true pK of 4.5. At neutral pH, the enzyme-inhibitor complex formation may induce a change in both the enzyme and the inhibitor structure; formation of the trypsin-proflavin complex, for instance, makes the inhibitor optically active.

Correlation among the proton charges and molecular masses of myosin subunits

The molecular masses and isoelectric points of myosin light and heavy chains were calculated from their known primary sequences and their respective distribution in a two‐dimensional graph is displayed. Implications for the electrophoretic study of myosin subunits are inferred from this analysis.

Étude thermodynamique de la dénaturation thermique réversible de la trypsine entre pH 1.0 et 3.4

Abstract A thermodynamic study of the thermodenaturation of bovine trypsin (EC 3.4.4.4) has been made by polarimetric and spectrophotometric measurements in the pH range from 1.0 to 3.4. The results show that a reversible equilibrium between a low-temperature conformation A and an high-temperature conformation D of trypsin exists. The increase in the half-transition temperature with pH may be due, as Pohl 5 suggested recently, to the ionization of two carboxylic groups of the protein. In the following relation, log K app ( pH , δ°) = log K T 0 (δ°) + log 1 + [H] K D 1 + [H] K A 2 Kapp is the experimental equilibrium constant, KTo is the true equilibrium constant between molecules A and D, the carboxylic groups of which are ionized, KD and KA are the ionization constants of these groups in the D and A molecules. A relatively good fit between the pH dependence of log Kapp and log {1 + ([H]/KD)/1 + ([H]/KA)}2 is obtained when pKA and pKD values are 1.0 and 4.6, respectively.

Note sur l'autoassociation de la trypsine a pH 4.8

Abstract The effect of protein concentration on some physicochemical properties of trypsin (EC 3.4.4.4) has been studied at pH 4.8, 10° and I = 0.3. It is shown that, under these experimental conditions, trypsin dimerization is a slow process. The results of light scattering indicate, moreover, that an isomerization step precedes the dimerization step, according to the following scheme: X→Y (1) and 2Y → 2 (2) The rate constant of the isomerization kinetics is about 1.6 · 10−2 min−1. This isomerization step proceeds without any significant change in the optical properties of the protein. The dimerization is reversed by protons (the protein is a monomer at pH 2.1). It is also inhibited by butylamine, a competitive inhibitor of trypsin; hence, it seems that only the monomeric form of the enzyme can bind the “substrate”.