An electron density map at 5.5 Å resolution has been computed for γ-chymotrypsin inhibited with toluenesulfonyl fluoride. Data were collected for the native enzyme crystals, for the inhibited enzyme tosyl-γCHT††Abbreviations used: CHT, chymotrypsin; tosyl-F, toluenesulfonyl fluoride; pipsyl-F, p-iodobenzenesulfonyl fluoride; PMMCMBS-, p-methoxy-m-chloromercuribenzenesulfonyl-; PMS-, phenylmethanesulfonyl. and for four heavy-atom derivatives prepared by soaking the crystals in p-iodophenylsulfonyl fluoride, p-methoxy-m-chloromercuribenzenesulfonyl fluoride, KI3 and K2HgI4. The shape of the molecule has been determined and the position of the active center located. The γCHT molecule has been compared with αCHT.

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11.0); (2) ionic nature of eluant (Na salts of PO4, F−, Cl−, SCN−, ClO4−, and CaCl2); and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PO4, F−, Cl−, SCN−, and ClO4−, and low (<0.003 m) Ca2+; (2) acidic proteins which elute at about equal moderate molarities of PO4 and F−, but do not elute with Ca2+ and usually not with Cl−; (3) neutral proteins, which elute with PO4, F−, and Cl−, but show a strong anion specificity, and do not elute with Ca2+ or SCN−. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.

The author would like to express his appreciation to Professor Ephraim Katchalski for his hospitality, to Professor Michael Sela for samples from his collection and for valuable discussion, to Professor Franz Sondheimer for the use of a spectrophotometer, and to Dr. Israel Schechter and Mr. Shmuel Shaltiel for valuable advice. Abbreviations used are: pTR, poly-L-tyrosyl ribonuclease; pApTR-L, lightly alanylated pTR, i.e., poly-DLalanyl-poly-L-tyrosyl ribonuclease; pApTR-H, heavily alanylated pTR; A(T,G)L, polv-DL-alanyl-poly(L-tyrosyl, L-glutamyl)-poly-L-lysine; poly-(L-tyrosyl, L-glutamyl)-poly-DL-alanyl-poly-L-lysine; R, ribonuclease; pAR, poly-DL-alanyl ribo-nuclease; and pTpAR, poly-Ltyrosyl-poly-DL-alanyl ribonuclease. The specific activity of I'a5 inhibitors was determined using standard solutions prepared by weighing out the solute (±0.2%). I125 activity was measured with a Packard automatic B-ray spectrometer. Enzymatic activity was assayed by the method of Hummel., Crystallographic studies: X-ray diffraction photographs were taken with a Buerger precession camera using CuKa radiation. The [hOt] and [hk0] data were collected to a resolution of approximately 4 A. Quartz calibrated photographs were used to determine the cell constants. The intensities of the reflections were determined with a Joyce-Loebl microdensitometer. E. coli K-13, a prototrophic isolate of strain K-12, and Hfr Hayes, a methionine requiring K-12 "relaxed" mutant, were used in the present experiments. Cells were grown in minimal medium buffered by 0.05 M triethanolamine at pH 8.2, supplemented with 0.5% sodium succinate and 0.2% casamino acids as sources of carbon as described previously.2 For the "step-down" experiment the enriched medium was prepared by adding 1% Difco Bacto-peptone to the minimal medium and readjusting the pH to 8.2.