No abstract

The relationship between the value of the nonlinear parameter B/A and the ratio of free water and bound water in a medium predominantly composed of water is examined. First, it is shown that in pure water the observed value of B/A can be expressed as (B/A)=vf(B/A)f +vb(B/A)b, where vf and vb are volume fractions of free and bound water, respectively. Next, a small dependency of the values (B/A)f and (B/A)b on temperature is noted. Then, the role of free and bound water in soft tissue is discussed in connection with the application of B/A analysis for medical diagnosis. Finally the additive property is examined for agarose gel.

The mono-complex formation of 2-thenoyltrifluoroacetone (HTTA) with nickel(II) and copper(II) ions was studied in aqueous solution at 25.0 °C and an ionic strength of 0.20 mol dm−3 by means of a stopped-flow method. The rate-[H+] profiles for the formation of these mono-complexes revealed that the rate-determining step of the complex formation interchanges depending upon hydrogen-ion concentration, as in the case of the Ni(II)–bpy complex. The rate constants of the mono-complex formation with TTA−, kf, and that with HTTA, kfh, were determined from the rate-[H+] profile in the region of the high hydrogen-ion concentration, where the pseudo-equilibrium of acid dissociation of the ligand was maintained during the complex formation; kf=(9.2±3.0)×102, kfh=(1.3±0.3)×10−1 mol−1 dm3 s−1 for the Ni(II)–TTA complex and kf=(2.0±1.2)×103, kfh=(5.0±0.9)×10−1 mol−1 dm3 s−1 for the Cu(II)–TTA complex. The deprotonation-rate constants of HTTA could be evaluated from the apparent rate constants of the complex formation in low hydrogen-ion concentration. The values thus obtained agreed well with that determined independently by a pH jump method.

Methyl N alpha-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for alpha-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2,3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1,2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by alpha-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl N alpha-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of alpha-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with Ki values which are generally smaller than the Km values for N alpha-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility necessary for catalysis.