The Mode of Protonation of Amides

The relative basicities of oxygen versus nitrogen in amides is a problem which has not been satisfactorily resolved. However, it might be anticipated from considerations of resonance and structural parameters that in strongly acidic solutions the proton will attach to oxygen rather than to nitrogen, to give [structure] I rather than [structure] II. It is generally recognized that amides are monoprotonated in 100 per cent sulfuric acid. For example, O'Brien and Niemann [1] reported i-factors of 2.0, 2.7, 2.0, 2.9, and 1.9 for benzamide, glycinamide, trichloroacetamide, benzoylglycinamide, and phthalimide, respectively, in this solvent. Similarly, in this study we have found the i-factors of N,N-dimethylformamide, N,N-dimethylacetamide, and acetamide to be 2.1, 2.1, and 2.0, respectively. Thus the problem is to locate the locus of protonation in singly protonated primary, secondary, and tertiary amides

A spectrophotometric study of blood group A-specific substance isolated from hog gastric mucosa

A study of the ultraviolet absorption spectra of preparations of blood group A-specific substance isolated from hog gastric mucin supports the view that group A substance, and probably group O substance, has little specific absorption in this spectral region and that other components account for most if not all of the specific absorption noted in the case of impure preparations. However, ultraviolet spectrophotometric analyses have been found to be particularly useful in following changes in composition brought about by the fractionation of group A substance preparations obtained from hog gastric mucin as well as in comparing preparations isolated by different methods and from different sources. Such analyses have also indicated that tyrosine and tryptophan, if indeed present in group A substance, are but minor constituents

A spectrophotometric study of the behavior of carbohydrates in seventy-nine per cent sulfuric acid

The spectral characteristics of solutions of eighteen representative monosaccharides in 79 per cent sulfuric acid have been determined for solutions maintained for 2 hours at 25° and 15 minutes at 100°. The use of these data for the identification of ketohexoses and 6-desoxyaldohexoses has been pointed out, as has their application to the quantitative determination of certain monosaccharides when present singly or in admixture with other monosaccharides, either as the monosaccharides or as components of oligosaccharides and polysaccharides

The competitive inhibition of of the urease-catalyzed hydrolysis of urea by phosphate

The urease-catalyzed hydrolysis of urea has been found to be competitively inhibited by phosphate at pH 7.0 and 25°. The Michaelis constant of the urea-urease system has been found to be approximately 0.003 M urea and the comparable constant defining the phosphate-urease system 0.035 M phosphate

A procedure for the determination of proteolytic activity

The difficulties introduced by the desire to maintain a constant pH during an enzyme-catalyzed hydrolysis of peptide-like substrates and at the same time to determine the extent of hydrolysis by an acid-base titration have been pointed out (1), but to date no completely satisfactory solution of the problem has been given. With those enzymes whose pH optima lie in the region between pH 7.5 to 8.5, e.g. trypsin and chymotrypsin, the poor buffering capacity of phosphate in this region prompted us, as it has others (2-5), to consider the use of organic amines whose pK’alpha values were near to or identical with the pH optimum of the enzyme being used. In the course of such studies it soon became evident that coincidental use of a suitable primary or secondary amine buffer system and a formol titration (1) would insure adequate buffering capacity with low buffer concentration during the hydrolysis and at the same time permit the final acid-base titration to be conducted under nearly ideal conditions. In this communication we shall limit the discussion to results obtained with chymotrypsin and specific acylated-a-amino acid amide substrates, since the application of the general method to other proteolytic enzymes and other types of substrates will be obvious

The hydrolysis of N-benzoyl-L-argininamide by crystalline trypsin

A reinvestigation of the kinetics of hydrolysis of N-benzoyl-n-argininamide by crystalline trypsin has led to the conclusion that the hydrolysis products enter into the over-all reaction as inhibitors

The use of ion exchange resins in the isolation of blodd group A-specific substance from hog gastric mucin

Ion exchange resins (1) have been used in the investigation of A substance hydrolysates (2), but apparently no attempt has been made to determine whether they can be profitably used in the isolation or purification of undegraded A substance [1] from sources such as hog gastric mucin. Studies along these lines are reported in this communication

The dependence of the specific activity of urease upon the apparent absolute enzyme concentration

If the activity of an enzyme preparation is determined under conditions in which a further increase in substrate concentration is without demonstrable effect, all other factors being held constant, it is ordinarily assumed that the specific activity of the enzyme, expressed in terms of arbitrary units per unit weight of the enzyme, is independent of the absolute enzyme concentration (1, 2). However, with urease solutions stabilized with hydrogen sulfide or cysteine (1) we have observed that the specific activity of a given urease preparation, when determined under the above conditions, increases with decreasing apparent enzyme concentration over a wide range of concentrations and that this increase in specific activity proceeds with a measurable velocity at temperatures above 15°. This phenomenon was observed with crude urease preparations, such as jack bean meal, and with two, three, and seven times recrystallized urease. Since little or no difference was observed in the behavior of three and seven times recrystallized urease, the data presented in this paper are limited to those obtained with thrice recrystallized preparations. Urease activity was determined by a modification of the procedure described by Van Slyke and Cullen (3). The precision of the modified procedure was ±2 to 3 per cent

The colorimetric determination of hexoses with carbazole

The optimum conditions for the calorimetric estimation of hexoses by reaction with carbazole in hot sulfuric acid solution have been determined and a convenient procedure, giving results with a precision of 2 to 5 per cent in the range of 50 to 150 γ of glucose, is described. The colors obtained with glucose, galactose, fructose, and mannose are not sufficiently distinctive to allow their ready differentiation and identification by spectral measurements. The significance of the ultraviolet spectra of heated and unheated sulfuric acid solutions of hexoses to the problem of estimation and identification of hexoses is discussed