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1. The effect of maltose was studied in porcine pancreatic amylase. At neutral p H 101, (29 mM) maltose produced with amylase a difference spectrum characteristic of the perturbation of tryptophan. The molar absorption Werence a t the maximum wavelength was AE~,,, = 1200.2. The difference spectrum appeared to be specific for maltose. Perturbation difference spectra measurements in 20°/, polyethylene glycol indicated that one tryptophyl side chain per mol amylase was involved in the interaction with maltose.3. The dissociation constant of the amylase * maltose complex calculated from the concentration dependence of the absorption difference a t 290 nm was Ks = 13 mM. Maltose inhibited amylase activity competitively and an inhibition constant of Ki = 25 mM was obtained, a similar value to that found spectrophotometrically. It is assumed that the tryptophyl side chain interacting with maltose may be involved in the binding of substrate by pancreatic amylase.It has been known for a long time that n-amylases of various origins split 0~-1,4-glucosidic bonds in a random manner which finally produces mainly maltose from starch, glycogen or dextrins (cf. Fischer and Stein [l]). So far, the action of the enzyme has been studied by analyzing the various products of the enzymatic process. Practically no information is available about the nature of side chains of pancreatic amylase which participate in the binding and hydrolysis of polysaccharide substrate. It was found with Bacillus subtilis amylase that oxidation of one tryptophyl side chain completely inactivated the enzyme [2], but did not alter its immunological properties [3]. Modification of several tyrosyl side chains in the bacterial enzyme with tetranitromethane or acetylimidazole also resulted in the loss of enzyme activityThe enzyme substrate interaction can be studied in some cases also by means of spectrophotometry, when the binding of the substrate or product to a chromophoric group of an enzyme causes changes in its environment. It was shown by Hayashi et al. [5] that the binding of substrate analogues to lysozyme produced difference spectra, which were characteristic of the perturbation of one tryptophyl side chain per mol enzyme [6]. A related phenomenon was observed by Benmouyal and Townbridge [7] with trypsin and chymotrypsin .

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About 50 % of the carbohydrete moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2 -5 glucosamines and 1 -2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodate oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence.