A note on the application of the colorimetric ninhydrin determination to studies of enzyme kinetics.

Although the validity of the calorimetric determination of single amino acids with ninhydrin, described by Harding and MacLean (l), is unquestioned, the failure of this determination for mixtures of amino acids, together with the observation that ninhydrin produces a color with ammonia (2) and with certain amines and amides (3), seems to have led to the neglect of this useful procedure. The discovery by Bergmann and his coworkers (4, 5) of simple synthetic substrates for proteolytic enzymes has provided a powerful tool for the study of enzyme kinetics, but the analytical methods commonly used have been the limiting factor, both in time and accuracy, in these studies. A determination of the absorption spectrum of the color developed with phenylalanine in the Harding-MacLean method with a Coleman junior spectrophotometer revealed a rather sharp maximum at 572 mp. Ninhydrin-pyridine-water blanks have a minimum absorption in this same region so that unchanged ninhydrin causes no interference with the determination. Although the color developed by quantities of Dr.,-phenylalanine up to 10 micromoles was found to follow Beer’s law when transmissions were measured at 572 rnp, to date a suitable blank has not been found, with the result that the straight line relating per cent transmission on a semilogarithmic scale to quantity of phenylalanine does not pass through the point corresponding to zero phenylalanine and 100 per cent transmission. The intersection of the calibration line with the 100 per cent transmission axis occurs at 0.3 micromole of phenylalanine. This value must be regarded as the present lower limit of the method. Harding and MacLean (1) did not apply the method to quantities of amino acids greater than 3.6 micromoles, but it has been found that the quantities of reagents used by these workers are adequate for amounts of phenylalanine up to 5 micromoles. The determinations were extended to 10 micromoles by simply doubling the quantities of reagents used; i.e., by using 2 ml. of 10 per cent pyridine and 2 ml. of 2 per cent ninhydrin. A series of determinations indicated that. quantities of phenylalanine between 0.5 and 10 micromoles could be estimated with an error not greater than