Table 1 of
Gross, Mol Vis 2000;
6:30-39.Table 1 of
Gross, Mol Vis 2000;
6:30-39.
Table 1. Test of protease activity in IRBP
About 20 ng of IRBP (n = 14) was incubated with succinylated casein substrate for 25 h at 37 °C. Succinylation blocks primary amines in the protein. Should there be protease activity in a tested protein preparation, the casein would be digested, exposing unblocked primary amines, which react with trinitrobenzenesulfonic acid and produce a colored product. Absorbance at 450 nm was measured before and after the incubation. The value for IRBP represents the mean ± the standard deviation. In this experiment, the IRBP sample used was R12+ [13]. These results are typical of those obtained with other human IRBP recombinant proteins. By t-test, the difference between A450 before and after incubation (0.0058) of the IRBP samples is not significant (p = 0.458), indicating no protease activity in this IRBP preparation. Trypsin, even at the lowest level tested of 0.12 ng, gave a detectable increase in absorbance (0.079) over the starting value. Buffer with substrate gave a small increase in absorbance of 0.012 after incubation.
A450 before A450 after
incubation incubation
--------------- ---------------
IRBP (n = 14) 0.0802 ± 0.0314 0.0860 ± 0.0297
3.90 ng Trypsin 0.031 0.491
1.95 ng Trypsin 0.026 0.336
0.980 ng Trypsin 0.029 0.299
0.488 ng Trypsin 0.030 0.260
0.244 ng Trypsin 0.029 0.185
0.122 ng Trypsin 0.033 0.112
Buffer (no protein) 0.037 0.049
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