细胞定位：Secreted.
Trypsin is a pancreatic serine protease with substrate specificity based upon positively charged lysine and arginine side chains. It is derived from a 34 kDa inactive precursor zymogen, trypsinogen, after enzymatic removal of an N-terminal 6-amino acid leader sequence resulting in the 23.8 kDa trypsin molecule. The optimum pH is 8.0. Trypsin is inhibited by organophosphorus compounds such as diisopropylfluorophosphate and natural inhibitors from pancreas. Soybean, lima bean, and egg white are also sources of natural inhibitors. Trypsin cleaves amide and ester bonds of Arg and Lys.
Uses: For tissue culture work. This product is typically used for protein sequencing, mapping and structure studies.
Technical Notes: A chromatographically purified, diafiltered, lyophilized powder that has been treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inhibit contaminating chymotryptic activity. Store at 2-8°C. PROTECT FROM MOISTURE.
Trypsin Assay
Method
One unit hydrolyzes 1 umole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at 25°C, pH 8.2, in the presence of 0.01 M calcium ion.
Reagents
0.046 M Tris⋅HCl buffer, pH 8.1 with 0.0115 M calcium chloride
0.01 M TAME (p-toluene-sulfonyl-L-arginine methyl ester)
0.001 N HCl
Enzyme
Dilute to a concentration of 10-20 ug/ml in 0.001 N HCl.
Procedure
Set spectrophotometer at 247 nm and 25°C.
Pipette into each cuvette as follows:
0.046 M Tris⋅HCl buffer, pH 8.1 2.6 ml
0.01 M TAME 0.3 ml
Incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration and establish a blank rate, if any. Add 0.1 ml diluted enzyme and record A247 for 3-4 minutes. Determine ΔA247 from initial linear portion of the curve. The reaction remains linear to an A247 of about 0.320. The reaction should be linear for at least three minutes. If this is not so repeat using less enzyme.
Calculation
where 540 is the extinction coefficient of p-toluenesulfonyl-L-arginine at 247 nm
1 TAME Unit = 19.2 USP/NF Units = 57.5 BAEE Units.
H2606324
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