SummaryThe molar ellipticity of the warfarin-albumin complex at 310 nm increases with pH from 6 to 9. This pH dependence runs parallel with that of the molar ellipticity of the albumin alone at 292 nm. The change in molar ellipticity with pH occurs in a smaller pH interval after addition of the physiological concentration of calcium ions. These findings give support to the assumption that the binding site for warfarin on the albumin molecule is affected by the neutral-to-base transition in the protein.Several authors have reported a pH dependent conformational change to occur in serum albumin around the physiological pH [1--7]. Zurawski and Foster [ 2] showed evidence for the existence of two conformational states in bovine serum albumin, the N form mainly occurring at the neutral pH and the B form at higher pH. They call this conformational change, therefore, the neutral-to-base or the N-B transition. Leonard et al. [ 1 ] found that the specific rotation of bovine and human serum albumin at 313.2 nm decreases between pH 7 and 9. This was interpreted as a change of the protein structure to another form when the pH was increased. This basic form is also favoured with increasing ionic strength. Nikkel and Foster [7] and Zurawski and Foster [2] later showed similar effects of pH on the conformation of bovine serum albumin using NMR spectroscopy to follow the chemically modified albumin molecule. Katz and Klotz [8] and Harmsen et al. [3] showed for bovine serum albumin that this N-B transition was effected by calcium ions.

The susceptibility of coumarins to alkaline hydrolysis, resulting in the formation of cis-coumarinate (and subsequently trans-coumarinate) polyanions is well known (Decker and Becker, 1922;Garrett et al., 1971; Mattoo, 1957; Bowden et al., 19611;Lippold and Garrett, 1971). For the most part, studies of substituent effects on the rates of alkaline hydrolysis of coumarins have been limited to derivatives with relatively weakly interacting functional groups. However, the coumarins of greatest economic interest are hydroxylated derivatives some of which are used as anticoagulants, fluorescent probes and optical components of mode-locked lasers (Shank et al., 1970; Trozzolo et al., 1974;Schulman and Rosenberg, 1979). The resistances of these substances to decomposition (Connors et al., 1979) under various solution conditions is therefore, of more than academic interest.The ionization of the hydroxy group which ought to play a dramatic role in the relative Iabilities of variously substituted hydroxycoumarins to attack by OH-, introduces an excellent opportunity to assess the relative importances of strong conjugative and electrostatic influences on the hydrolysis of coumarins.In the present note we consider the influence of the position of the hydroxy group on the second-order 'rate constants for the hydrolysis of 3-, 4-and 7-hydroxycoumarin and some related anticoagulants-warfarin, acenocoumarol and phenprocoumonthe drugs of choice in the U.S.A., The Netherlands and the Federal Republic of Germany, respectively. Because the hydroxy groups of all the compounds studied are fully ionized at the pH where the hydrolyses were studied * To whom correspondence should be addressed. ' The value of pK, -7.12fO.05 at 22OC was recently reported for the 3-isomer (Wolfbeis, 1981).0378-5173/82/0000-0000/82.75 D

The free SH group in bovine serum albumin has been modified by covalent coupling with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-2,4-dinitrophenol