322 / Biochemistry Amino acids, continued

Fig. 10.4 (continued) tide bond cleavage. In the cysteine (and also serine and threonine) proteases, the nucleophile is the protease type amino acid (in this case cysteine) which forms a covalent bond with the carbon atom of the bond to be cleaved (covalent catalysis) in contrast to the metalloprotei-nases and aspartic proteases which use an activated water molecule to attack the carbon atom to be cleaved (noncovalent catalysis). In covalent catalysis, a nearby histidine residue normally functions as a base to activate the mechanism, whereas in noncovalent catalysis, the protease type serves as an acid and base, with an ancillary histidine (aspartate proteases) or aspartate or glutamate residue acting as the nucleophile (Fig. 8.2b) (Modified from Fig. 9.18 in Berg., et al., Biochemistry, 5th Ed. 2002, W.H. Freeman Co., New York)...

The isotope dilution technique has been employed for Ihe determination of about thirty elements in a variety of matrix materials, Isotopic dilution procedures have also been most widely used for the determination of compounds of interest in organic chemistry and biochemistry, Thus, methods have been developed for the determination of such diverse substances as vitamin D, vitamin B12, sucrose, insulin, penicillin, various amino acids, corticosterone, various alcohols, and thyroxine. Isotope dilution analysis has experienced less widespread application since the advent of activation methods. Continued use of the procedure can be expected, however, because of the relative simplicity of the equipment required. In addition, isotope dilution is often applicable where activation analysis is not. 

In the 1950s, W. H. Stein and S. Moore conducted pioneering work at Rockefeller Institute to understand the structure of the enzyme ribonuclease, for which they were awarded the Nobel Prize in 1972. Continual need to measure amino acids led them to explore amino acid separations by ion exchange. By 1958, their design led to the first commercial automated amino acid analyzer— a revolutionary advance for biochemistry. 

When Severo Ochoa left the Biochemistry Department at New York University I had to search for another mentor and I was fortunate to be able to continue my graduate work with Albert Keston. While developing the isotope derivative method for amino acid assay we showed for the first time that hydroxyproline was uniquely present in collagen. This observation made a great impression on me. Furthermore, I had the good fortune while at New York University Medical School to meet and to get to know the late Joseph Bunim. It was from him that I learned about the role of collagen in connective tissue and also about collagen diseases. 

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