Carboxypeptidase Y 317
nmol of carboxypeptidase Y in 5-10 lxL of 0.1M pyridine acetate buffer,
pH 5.6, thoroughly mix, incubate at room temperature, and remove
25-ktLaliquots at T= 1,2, 5, 10, 20, 30, and 60 min. Add 5 l.tLof glacial
acetic acid to each sample to stop the reaction. Samples are then fro-
zen, lyophilized (pyridine acetate is volatile), and then subjected to
amino acid analysis. The small amount of SDS in each sample applied
to the amino acid analysis does not interfere with the elution profile or
affect the integrity of the machine. The sampling times indicated here
should be appropriate for most proteins. However, should the C-termi-
nal sequence be such that a number of slowly released amino acids are
present, then the experiment may have to be repeated using longer
incubation times or a higher enzyme-to-substrate ratio.
If analyzing peptides, SDS can be omitted from the incubation buffer,
since the C-terminal should be readily accessible. In addition, the time
intervals for sampling can be reduced, since the rate of appearance of
free amino acids will be faster than that for proteins. Use T = 0, 1/2, 1,
2, 5, 10, 15, and 20 min. A graph is plotted showing the amount of each
amino acid released with time and the C-terminal sequence deduced
from the relative rate of release of each amino acid (see, e.g., ref. 16).
References- Neurath, H. (1960) Carboxypeptidases A and B, in The Enzymes, vol. 4 (Boyer,
P. D., Lardy, H., and Myrb~ick, K., eds.), Academic, New York, pp. 11-36. - Sprossler, B., Heilmann, H. D., Grampp, E., and Uhlig, H. (1971) Carboxypepti-
dase C from orange leaves. Hoppe-Seyler's Z. Physiol. Chem. 352, 1524-1530. - Hayashi, R., Moore, S., and Stein, W. H. (1973) Carboxypeptidase from yeast.
J. Biol. Chem. 248, 2296-2302. - Johansen, J. T., Breddam, K., and Ottesen, M. (1976) Isolation of carboxypep-
tidase Y by affinity chromatography. Carlsberg Res. Commun. 41, 1-14. - Hayashi, R., Bai, Y., and Hata, T. (1975) Kinetic studies of carboxypeptidase
Y. J. Biochem. 77, 69-79. - Breddam, K. and Ottesen, M. (1987) Determination of C-terminal sequences
by digestion with serine carboxypeptidases. The influence of enzyme specific-
ity. Carslberg Res. Commun. 52, 55-63. - Hayashi, R., Bai, Y., and Hata, T. (1975) Further confirmation of carboxypep-
tidase Y as a metal free enzyme having a reactive serine residue. J. Biochem.
77, 1313-1318. - Hayashi, R., Moore, S., and Stein, W. H. (1973) Serine at the active centre of
yeast carboxypeptidase. J. Biol. Chem. 248, 8366-8369. - Hayashi, R., Bai, Y., and Hata, T. (1975) Evidence for an essential histidine in
carboxypeptidase Y. J. Biol. Chem. 250, 5221-5226.