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By Nathan O. Kaplan, Sidney P. Colowick, C. H.W. Hirs, Serge N. Timasheff
The significantly acclaimed laboratory typical, Methods in Enzymology, is likely one of the so much hugely revered courses within the box of biochemistry. given that 1955, every one quantity has been eagerly awaited, often consulted, and praised via researchers and reviewers alike. The sequence includes a lot fabric nonetheless appropriate this day - actually a vital booklet for researchers in all fields of existence sciences
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Extra info for Enzyme Structure, Part B
Example text
Adsorption to the column packing) are more likely to be manifest. ~2R. W. Zumwalt,K. Kuo, and C. W. Gehrke, J. Chromatogr. 57, 193 (1971). [4] D e t e r m i n a t i o n of T r y p t o p h a n 1 B y TF~-YUNG LIU As the precision of analytical methods for the determination of amino acids has increased, the limiting factor in determining the amino acid composition of a protein has become the extent to which the composition of the hydrolyzate is a true reflection of the composition of the parent protein.
32W. J. McBride and J. D. Klingman, in "Lectures on Gas Chromatography" (L. R. Mattick and H. A. ), p. 25. Plenum, New York, 1967. a~K. Blau, in "Biomedical Applications of Gas Chromatography" (H. A. ), p. 1. Plenum, New York, 1968. 40 AMINO ACID ANALYSIS AND RELATED PROCEDURES [3] determined as their amino acid counterparts as they are hydrolyzed in the commonly used esterification procedures. Of the dozens of derivatives that have been studied, two appear superior. They are the N-trifluoroacetyl n-butyl esters, ~',3~ and the trimethylsilyl derivatives26 N-Trifluoroacetyl n-Butyl Esters The formation of the derivative is effected in two steps using nbutanol and then trifluoroacetie anhydride (TFAA) as follows: RCH(NH~)COOH C4HgOH/HC1 TFAA/CH2Ch 100°/15 rain 150°/5 mln ~ RCH(NH2)COOC4Hg"HCI , RCH(NHCOCF3)COOC,H9 (1) The butyl esters are superior to the corresponding methyl esters because the latter are so volatile that some are subject to serious losses during the workupY ,3s The problems of poor solubility of amino acids in butanol and slow rate of ester formation have been overcome by ultrasonic mixing of the amino acid suspension in n-butanol which is made 3 N in HC1, prepared by bubbling dry acid into the alcohol.
The recovery of tryptophan from a mixture of tryptophan and bovine ribonuclease A I0 - LIJ *1 J, 8 I'0 n,- o. J 0 0¢ bJ 0. Ul hi :3 t~J Q: Z 1" 00 I-0.. >(E l-- I 24 HYDROLYSIS l 48 TIME (HOURS} I 72 Fx~. 2. {}, see Table II). 3 100 a Values are expressed as percentage of theoretical values. Duplicate analyses by ion-exchange chromatography were performed on 0-, 22-, 48-, and 72-hour hydrolyzates. Averages of the two analyses are presented. For computation of the percentage of recovery, the zero time value (sample without heating at 110°) was used as 100%.