VOL. 51, 1964 BIOCHEMISTRY: T. VISWANATHA 1117 as it rises in b to enter c above the midline, etc. It can be easily seen that a similar oscillation about the midline takes place in the rotating fluid annulus of Figure 4a and in the more general situation of Figure 4b. These oscillations can be made quite small by adjusting the density of the buffer to match the particle density and by speeding up the rate of buffer flow to shorten the period of settling. The principle of eliminating thermal convection in continuous flow electrophoresis presented here can be implemented experimentally in a variety of ways. The various methods of approach will be published in this journal. Summary.-The process of generation of thermal convection is considered with an emphasis on two special cases: (1) a thin fluid sheath between two parallel vertical walls with a horizontal temperature gradient maintained in the fluid; (2) a thin fluid sheath between two parallel horizontal plates with a vertical temperature gradient maintained in the fluid to obtain a density decrease with depth. The patterns of thermal convection are described, and it is shown how both types of convection can be inhibited by allowing the fluid sheath to flow through a closed or a meandering channel in such a fashion that the relative orientation between the gravitational field and the convection pattern is periodically reversed. * This work has been aided by a grant from the Office of Naval Research. Lord Rayleigh, Phil. Mag., 32, 529-546 (1916). 2 Jacob, M., Heat Transfer (Wiley, 1949), vol. 1. Sanders, V., "Laminar free convection between smooth vertical parallel plates," thesis, University of California (Los Angeles), 1962. 4 B6nard, H., Ann. Chim. Phys., 23, 62-144 (1901). 'Liu, Chen-Ya, "Natural convection heat transfer in long horizontal cylindrical annuli," thesis, New York Univ., 1961. 6 Hjertkn, S., Arkiv Kemi, 13, 151 (1958). 7 Kolin, A., these PROCEEDINGS, 46, 509 (1960). 8 Kolin, A., J. Chem. Phys., 22, 1628 (1954). 9 Kolin, A., Biochim. et Biophys. Acta, 32, 538 (1959). 10 Kolin, A., J. Appl. Phys., 25, 1442 (1954). 1Kolin, A., and P. Cox, these PROCEEDINGS, in press. STUDIES ON THE ACTIVE SITE OF CHYMOTRYPSIN* BY T. VISWANATHA INSTITUTE OF MOLECULAR BIOLOGY, UNIVERSITY OF OREGON, EUGENE Communicated by V. Boekelheide, April 6, 1964 Studies were undertaken to investigate the nature of the amino acids, especially of the serine residue, associated with the phosphopeptides of DIP-chymotrypsin,l DIP-trypsin,2 and DIP-subtilisin (Novo).3 a-Chymotrypsin was first investigated. In a typical experiment, 2.5 gm of 3 X recrystallized a-chymotrypsin was converted to its DIP-derivative by treat- ment with DFP at pH 8.0. After the removal of excess DFP by extensive dialysis against 0.001 N HC1, the DIP-protein was subjected to enzymatic degradation at 370C in the following order: the pepsin at pH 2.10 (8 hr); chymotrypsin and tryp- sin at pH 8.0 (8 hr); carboxypeptidases A and B at pH 7.80 (8 hr); and finally with Downloaded by guest on September 28, 2021 1118 BIOCHEMISTRY: 7'. VJSWANATHA PROC. N. A. S. leucylaininopeptidase at pH 8.0 (8 hr) in the presence of 0.005 AI MgC12. The total amount of each enzyme used was 1-1.5 parts per 1000 parts of DIP-protein. The enzymatic digest of the DIP-protein was adjusted to pH 4.0 and taken to dryness with the aid of a Rinco flash evaporator. The residue so obtained was refluxed with 25 ml of redistilled HCl for 20 min. The hydrolysate was taken to dryness under vacuum. The purification and the properties of the phosphopep- tides are described in the flow sheet. FLOW SHEET SHOWING THE PURIFICATION OF PHOSPHOPEPTIDES Hydrolysis of the Enzyme Digest of DIP-Chymotrypqin Remarks Dowex 50 (H+) Chromatography 1. Acidiefraction plus water washings 1. This fraction contains phosphopeptides and small amounts of aspartic acid and glu- Rechromatography on 150-cm long column tamic acid. Recovery of bound phosphorus of the amino acid analyzer; elution with 0.2 50% based on phosphate determination. N Na citrate buffer, pH 3.25, temp 500C. It reduces alkaline triphenyl tetrazolium Procedure the same as Spackman et al.4 chloride (TPTZ). 2. Fraction appearing at 40-60 ml effluent volume 2. This fraction reduces alkaline TPTZ; it re- collected and desalted solves into 3 components as shown in Fig. la; amino acid composition AsP: Ser-P: Rechromatography of material corresponding Gly: Glu: 1:2.5:1:1; Total nitrogen to peaks I & II (Fig. la) on 150-cm long present in an aliquot of sample 90 ug; column; conditions same as above except nitrogen recovered in the form of amino temp 30°C. acids in the acid hydrolysate of an identical aliquot of the sample 42 pg; material in Peak III analyses for Ser, ornithine, and arginine (see text). 3. Acidic comrnpornents (Fi. lb) 3. Component A reduces alkaline TPTZ; A i amino acid composition Asp, (Ser-P)2 Gly, A B C X2 (see text for details). Rechromatography4 on 50-cm medium column, 0.38 N Na citrate buffer, pH 4.26 (Fig. lc) 4. Al A2 4. Al - (Asp, Ser-P, Gly, X). A2 - (Ser, X). See text. The desalting of all the phosphopeptide fractions (2, 3, and 4, flow sheet) prior to chromatography was achieved by acidification with 2 N HC1 followed by extraction with ether to remove the citric acid formed. The ether-extracted material was taken to dryness and was then treated with 10 ml of absolute ethanol. After the removal of the precipitated NaCl, the alcohol solution containing the phospho- peptides was taken to dryness. The observed discrepancy between the total nitrogen determined according to the procedure of Lanni et al.5 and the nitrogen recovered in the form of amino acids from the phosphopeptide fraction 2 (flow sheet) prompted an analysis of the materialin peak III in Figure la. This material was isolated and was analyzed for its amino acid composition. An analysis of the acid hydrolysate of this material showed the presence of serine; ornithine and arginine were found upon treatment of the hydrolysate with ammonia. Serine and the basic amino acids were present in equimolar amounts. Downloaded by guest on September 28, 2021 VOL. 51, 1964 BIOCHEMISTRY: T. VISWANATHA 1119 .8 _ - .-(a) a.4_._ .. I- 00 27 47 67 .87 107 127 147 167 187 207 227 247 267 ° .6_ r (b) .2 , . -. z 0 26 46 66 86 106 126 146 166 I.6 _ .Ai ¢.4(C ) A2 0*° .2 _- . 0 A 0 23 43 63 83 103 123 143 163 183 203 223 243 263 28 EFFLUENT VOLUME (m8 FIG. 1.-(a) A typical chromatogram of the phosphopeptide fraction (2, flow sheet) on a 50-cm medium column. 0.38 N Na citrate buffer, tempr 3000, pH 4.26. (b) Rechromatography of the acidic material in phosphopeptide fraction (2, flow sheet) on 150-cm long column, 0.2 N Na citrate buffer, temp 300C, pH 3.25. (c) Chromatogram of component A (b) on 50-cm medium column. (All analyses were made immediately after the isolation of the fractions mentioned.) Since the above results suggested the presence of an unusual arginine residue in the phosphopeptide, further chromatographic purification of the phosphopeptides was accomplished leading to the isolation of component A (3, flow sheet). Phosphate and amino acid analysis of 16-hr acid hydrolysate of Component A showed the presence of P, Asp, Ser, and Gly in the ratio 2:1:2: 1. An ammonia peak with an accompanying shoulder was observed. No basic amino acids were noticed. How- ever, treatment of component A with 2 N ammonium hydroxide resulted in the release of arginine in amounts equivalent to the serine present. The same amount of arginine was found in the acid hydrolysate of the ammonia-treated component A, suggesting that all of arginine present was released free from the rest of the com- ponent A upon ammonia treatment. Thus, the amino acid composition of com- ponent A appears to be [Asp, (Ser-P)2gly, X2], X6 being the source of arginine. Treatment of 16-hr acid hydrolysate of component A with 3 N ammonium hy- droxide and analysis showed the presence of ornithine in addition to the amino acids mentioned earlier. Hydrolysis of component A with HCl in the presence of HCl04 led to the formation of glutamic acid and pyrrolidone carboxylic acid, in addition to the amino acids observed in the absence of HCl04. These results are consistent with X being an unusual arginine residue. Aspartic acid and serine were found to occupy N-terminal position from analysis of component A treated with dinitrofluorobenzene, suggesting component A is a mixture of two phosphopeptides. Indeed, on rechromatography of component A on 50-cm medium column, it resolves into two substances, Al and A2 (Fig. lc). The substance A2 results presumably due to dephosphorylation of one of the phosphopeptides present in component A, during the desalting procedure employed. It has also been observed earlier (peak III, Fig. la), and its amino acid composition is (Ser, X). The phosphopeptide Downloaded by guest on September 28, 2021 1120 BIOCHEMISTRY: T. VISWANATHA PROC. N. A. S. (Ser, X, P) which is the source of A2, arises from the degradation of Al during the acid hydrolysis employed in the preparation of phosphopeptides. The appearance of arginine following ammonia treatment of 3 X chromatographed component A (only the acidic fraction being used in each rechromnatography), as well as the close stoichiometric relationship observed between arginine and the other amino acids present in component A, suggest the presence of "an activated guanido group" in the chymotrypsin.