A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of... more A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of short-chain alkyl isocyanates. The procedure is based upon measurement of urea derivatives formed from reaction of the isocyanates with amines. Gas chromatographic and thin-layer chromatographic separations of a number of urea derivatives are described. Application of the method is demonstrated in studies of the decomposition of and urea formation from isocyanates in aqueous solutions.
Biochemical and Biophysical Research Communications, 1974
The reactivity of trypsinogen toward p-nitrophenyl-p'-guanidinobenzoate is increased when disulfi... more The reactivity of trypsinogen toward p-nitrophenyl-p'-guanidinobenzoate is increased when disulfide 179-203 is selectively reduced and converted to the S-aminoethyl-(179,203) derivative. The reaction of the modified zymogen and the active site titrant is characterized by an initial rapid rate (6.6 x lO-3sec-I) and a postburst steady state rate (I.5 x lO-4sec'1). The steady state rate is the same as that found with trypsinogen and trypsin. The enhanced rate of acylation of disulfide-modified trypsinogen must be associated with a change in conformation of groups at the specificity pocket, causing an increase in the affinity for active-site directed reagents. Activation of trypsinogen by the physiological activator enterokinase or by autocatalysis produces a limited proteolysis of the zymogen with cleavage of Lys(6)-lle(7) and release of the amino-terminal hexapeptide (1). A small change in the protein conformation generates the structure of the active site of trypsin. However, recent studies indicate that trypsinogen also displays a low intrinsic activity. Kay and Kassell found that trypsinogen slowly catalyzed its own activation at pH 8.1 (2). Recently, Neurath and associates reported a low catalytic activity of trypsinogen toward diisopropyl phosphofluoridate and Gdn-Bz-ONp ~ (3,4,5) and they also noted an inactivation of the zymogen by methane sulfonyl fluoride (6). A specific complex of trypsinogen and the basic pancreatic proteinase inhibitor has been observed in studies by Keil-Dluoha and Keil (7,8). Furthermore, several other zymogens, including chymotrypsinogen (3,5), procarboxypeptidase A (9), and pepsinogen The abbreviations used are: Gdn-Bz-ONp, p-nitrophenyl-p'-guanidino benzoate; (AE)2-trypsinogen, trypsinogen with disulfide 179-203 reduced and converted to the S-aminoethyl derivative; (AE)2-trypsin, S-aminoethyltrypsin produced on activation of the modified zymogen.
EXPERIMENTAL Materials. Trypsin (2 X crystallized), t.rypsinogen (1 X crystallized), and chymotry... more EXPERIMENTAL Materials. Trypsin (2 X crystallized), t.rypsinogen (1 X crystallized), and chymotrypsinogen A were purchased from Worthington Biochemical 377 Copyright 0 1971 by Academic Press, Inc. 411 rights of reproduction in any form reserved. 37s KNIGHTS AND LIGHT Corp. Soybean trypsin inhibitor (5 X crystallized) was a special grade from Gallard Schlesinger Chemical Mfg. Corp. Sepharose-4B was a product of Pharmacia Fine Chemicals Inc. Cyanogen bromide was from Pierce Chemical Co. N-Benzoyl-Larginine ethyl ester, N-benzoyl-m-arginine-pnitroanilide, N-acetyl-L-tyrosine ethyl ester, and ultrapure urea were purchased from Schwarz/ Mann. Concentrated urea solutions were deionized on a mixed-bed ion-exchange resin, Amberlite type MB-3, obtained from Mallinckrodt Chemical Works. The p-nitrophenyl-p'-guanidinobenzoate was a product of Cycle Chemical Corp. Protein concentrations.
A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of... more A gas chromatographic procedure is described for the quantitative analysis of dilute solutions of short-chain alkyl isocyanates. The procedure is based upon measurement of urea derivatives formed from reaction of the isocyanates with amines. Gas chromatographic and thin-layer chromatographic separations of a number of urea derivatives are described. Application of the method is demonstrated in studies of the decomposition of and urea formation from isocyanates in aqueous solutions.
Biochemical and Biophysical Research Communications, 1974
The reactivity of trypsinogen toward p-nitrophenyl-p'-guanidinobenzoate is increased when disulfi... more The reactivity of trypsinogen toward p-nitrophenyl-p'-guanidinobenzoate is increased when disulfide 179-203 is selectively reduced and converted to the S-aminoethyl-(179,203) derivative. The reaction of the modified zymogen and the active site titrant is characterized by an initial rapid rate (6.6 x lO-3sec-I) and a postburst steady state rate (I.5 x lO-4sec'1). The steady state rate is the same as that found with trypsinogen and trypsin. The enhanced rate of acylation of disulfide-modified trypsinogen must be associated with a change in conformation of groups at the specificity pocket, causing an increase in the affinity for active-site directed reagents. Activation of trypsinogen by the physiological activator enterokinase or by autocatalysis produces a limited proteolysis of the zymogen with cleavage of Lys(6)-lle(7) and release of the amino-terminal hexapeptide (1). A small change in the protein conformation generates the structure of the active site of trypsin. However, recent studies indicate that trypsinogen also displays a low intrinsic activity. Kay and Kassell found that trypsinogen slowly catalyzed its own activation at pH 8.1 (2). Recently, Neurath and associates reported a low catalytic activity of trypsinogen toward diisopropyl phosphofluoridate and Gdn-Bz-ONp ~ (3,4,5) and they also noted an inactivation of the zymogen by methane sulfonyl fluoride (6). A specific complex of trypsinogen and the basic pancreatic proteinase inhibitor has been observed in studies by Keil-Dluoha and Keil (7,8). Furthermore, several other zymogens, including chymotrypsinogen (3,5), procarboxypeptidase A (9), and pepsinogen The abbreviations used are: Gdn-Bz-ONp, p-nitrophenyl-p'-guanidino benzoate; (AE)2-trypsinogen, trypsinogen with disulfide 179-203 reduced and converted to the S-aminoethyl derivative; (AE)2-trypsin, S-aminoethyltrypsin produced on activation of the modified zymogen.
EXPERIMENTAL Materials. Trypsin (2 X crystallized), t.rypsinogen (1 X crystallized), and chymotry... more EXPERIMENTAL Materials. Trypsin (2 X crystallized), t.rypsinogen (1 X crystallized), and chymotrypsinogen A were purchased from Worthington Biochemical 377 Copyright 0 1971 by Academic Press, Inc. 411 rights of reproduction in any form reserved. 37s KNIGHTS AND LIGHT Corp. Soybean trypsin inhibitor (5 X crystallized) was a special grade from Gallard Schlesinger Chemical Mfg. Corp. Sepharose-4B was a product of Pharmacia Fine Chemicals Inc. Cyanogen bromide was from Pierce Chemical Co. N-Benzoyl-Larginine ethyl ester, N-benzoyl-m-arginine-pnitroanilide, N-acetyl-L-tyrosine ethyl ester, and ultrapure urea were purchased from Schwarz/ Mann. Concentrated urea solutions were deionized on a mixed-bed ion-exchange resin, Amberlite type MB-3, obtained from Mallinckrodt Chemical Works. The p-nitrophenyl-p'-guanidinobenzoate was a product of Cycle Chemical Corp. Protein concentrations.
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