HPLC‐analysis of the reaction products of a series of 4‐methylumbelliferyl glycosides from cello‐oligosaccharides, used as substrates of a cellobiohydrolase from Trichoderma reesei, proves the lack of specificity for terminal cellobiosyl groups. Also, different reaction patterns are observed for this CBHI and for an endocellulase, when acting on these same substrates. 4‐Methylumbelliferyl β‐D‐lactoside is an unexpected substrate for CBHI, yielding only lactose and phenol as reaction products. The binding characteristics of p‐nitrobenzyl 1‐thio‐β‐D‐lactoside for this enzyme are determined by a dia‐filtration technique, yielding 1 binding site and an association constant of 4.0 × 104 M−1.

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The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

The binding of 4-methylumbelliferyl per-N-acetyl-chitooligosaccharides, MeUmbGlcNAc (I), MeUmb(GlcNAc)z (11) and MeUmb(GlcNAc)s (111) to wheat germ agglutinin, was studied by equilibrium dialysis, difference absorption spectrometry and extrinsic fluorescence quenching [Privat, J. P., FEBS Lett. 46, 229-2321 titration at a fixed wavelength or above 370 nm. The change in optical properties of the MeUmb group upon binding of the glycosides to the agglutinin depends on the length of the carbohydrate chain. An intense and carbohydrate-specific difference absorption spectrum was observed with I (-Ae at 316 nm equal to 2770 M-' cm-I), a weaker one with I1 ( -A s at 316 nm = 1070 M-' cm-'), but none was observed with 111; the fluorescence quenching is 100% for compounds I and I1 and 44% for compound 111. As determined with I, using equilibrium dialysis and difference absorption spectrometry, there are two identical and independent binding sites per subunit. For I the value of the association constant is independent of the experimental method or the saturation range used. It has long been suggested [1,2] (for a recent review see [3]) that wheat germ agglutinin possesses an extended binding site, possibly accomodating two or three GlcNAc residues. As determined [4,5] by the enhancement of intrinsic tryptophan fluorescence, the value of the association constants for the (GlcNAc), series increases with the length of these oligosaccharides. With the corresponding fluorescent 4-methylumbelliferyl glycosides MeUmb(GlcNAc),, which were