Anionic trypsin

The polar cavity of Asp-102 also includes two buried water molecules, as well as backbone amides of Ala-56 and His-57. In about 200 SP, all but three have this Ser-214 residue. It is solvent inaccessible, and its OG forms a hydrogen bond to Asp-102. In an extensive experimental study by McGrath et al.(1992), Ser-214 has been mutated to Ala, Glu and Lys in rat anionic trypsin and both kinetics and crystallography were employed to study the... 

Figure 4.9 Anion-exchange liquid chromatography of acidic proteins. Column, Asahipak ES502N eluent, 20 min linear gradient of sodium chloride from 0 to 500 pM in 50 mM bis-tris/HCl buffer pH 7.0 flow rate, 1ml min-1 detection, UV 280 nm. Peaks-. 1, conalbumin (Mr 77000-88000, pi 6.0-6.8) 2, ovalbumin (Mt 45000, pi 4.6) 3, trypsin inhibitor (Mr 8000, pi 4.5) and 4, / -lactoglobulin (Mr 18400, pi 5.1).

Analysis of the growth promoter 4-hydroxy-3-nitrophenylarsonic acid (roxarsone) has been investigated by Dean et al. [78]. Tissue, sampled from chickens with a roxarsone supplemented diet was digested using a trypsin enzymolysis technique. Anion-exchange chromatography was used to per-... 

K8. Kimland, M., Russick, C., Marks, W. H., and Borgstrom, A., Immunoreactive anionic and cationic trypsin in human serum. Clin. Chem. Acta 184, 31—46 (1989). 

There is evidence that protein structures are also responsible for cell cohesion in nonpalmo-plantar stratum corneum. When punch biopsies of normal human gluteal skin were incubated in a buffer containing a mixture of the zwitterionic surfactant /V,/V,-dimethyldodecylamine and the anionic surfactant sodium dodecyl sulfate,11 there was dissociation of cells in the stratum corneum but not in the rest of the epidermis. The cell dissociation took place only in the presence of EDTA and was inhibited by the serine protease inhibitor aprotinin.12 Suzuki et al.13,14 presented evidence that spontaneous cell dissociation in nonpalmo-plantar stratum corneum could be inhibited by a combination of inhibitors of trypsin-like and chymotrypsin-like enzymes. Thus, nonpalmo-plantar stratum corneum contains endogenous proteases that mediate cell dissociation. 

Waters Q-8HR (strong anion exchange, 8 (jl particles) and DEAE-8HR(weak anion exchange, 8 fi particles) Pentosan polysulfate, dextran sulfate /3-lactoglobulins A and B, a-lactalbumin, soybean trypsin inhibitor (STI) 38... 

Note that proteolysis in conjunction with ion-exchangers may be used in special cases. The pH used with anion exchangers (typically around 8) may induce the activation of proteases such as trypsin-like proteases as well as plasmin and kallikrein when traces are present (these come from animal sera). In this case DEAE and Q, ion-exchangers should therefore be avoided as the first separation chromatographic column. 

Figure 7 Effect of salt concentration on migration velocity of DMSO, bovine carbonic anhydrase (BCA), a-lactalbumin (a-LAC), soybean trypsin inhibitor (STI), and ovalbumin (OVA). Conditions column 50 pm i.d. x 340/260 mm, packed with strong anion exchanger mobile phase, 50, 100, and 200 mM NaCI in 5 mM phosphate buffer, pH 7.0 applied voltage, -1 5 kV injection, 6 s at-8 kV UV detection, 200 nm. (Reprinted with permission from Ref. 48, copyright 2000, American Chemical Society.)...

Figure 1. SDS-PAGG analysis of alkaline-dissolved Bacillus thuringiensis subspecies kurstaki (BTK) and israelensis (BTI) -endotoxin at 25 yg per track (1) BTK -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.), (2) BTI 6-endotoxin from Sandoz Inc., (3) BTI (Sandoz) -endotoxin purified by DEAE-anion exchange chromatography, (4) percipitate formed after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (5) soluble fraction after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (6) BTI (Sandoz) -endotoxin purified by Sephadex G-75 gel filtration chromatography at Rf 1.35, (7) at Rf 1.58, and (8) BTI strain IFC-1 -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.). S, molecular weights as indicated X1000 for bovine serum albumin (BSA), ovalbumin (OA), trypsin, and myoglobin. Reproduced with permission from Ref. 29. Copyright 1984, Academic Press, Inc.

Titration curves of trypsin were obtained under a variety of conditions by Duke et al. (1952). The most noteworthy feature is a specific effect of calcium, which displaces the acid part of the titration curve to lower pH, and decreases the total number of groups which are titrated between pH 6 to 9. It is likely that the groups titrated between pH 6 and 9 in the absence of Ca " are a-amino groups, produced by self-digestion of the enzyme. The effect of Ca" " thus appears to result from a complex with the carboxyl groups of the protein, which stabilizes the anionic form of these groups so as to produce the displacement of the acid part of the titration curve. This complex is more resistant to self-digestion than the enzyme alone. 

TRY-1 is also described as cationic and TRY-2 as anionic because of their differing electrophoretic mobdity the cationic form predominates and is the better-documented enzyme. TRY-1 and TRY-2 have molecular weights of 25,800 and 22,900 and pi values of 4.6 to 6.5 and greater than 6.5, respectively. TRY-2 differs from TRY-1 in that it rapidly undergoes autolysis at neutral or alkaline pH values and Ca " does not stabilize it against autolysis. Because the two trypsins show little immunological cross-reactivity, specific immunoassay of each of them is possible. 

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