Functional study of interleukin-1b using various site specific chemical modifcation reagent

1. Chemical modifications : An application to interleukin-1  , a structural and functional approach.

2. Reactive amino groups in IL-1  (purple regions) by NHS-biotinylation studies Lysines 93,94 Lysine 103* Alanine 1 Journal of Biological chemistry (1989) 264:17691-17697

3. Reactive amino acid side chains that react with Acrylodan (A cystiene specific and environment sensitive reagent) Journal of Biological Chemistry (1992) 267:3122-3128

4. All chemical modifications introduced do not have any effects on IL-1  bioactivity. (Thymocytes proliferation assays & receptor binding measurements)

5. Isolation of EDC (water soluble carbodiimide) modified rIL-1  using TSKSP5PW Modified protein retains native pI

6. EKC peptides from rIL-1 

7. Native proteolysis of IL-1  using Endo-Lys-C (Native IL-1  Vs X-linked IL-1 

8. 1-88, NYPK and 93-150

10. EKC treatment post denaturation

14. Definition of site of carbodiimide reaction by Mass Spec. (FAB) after V8 (Endo-Glu-C) treatment

15. Location of salt bridge where carbodiimide crosslinkage was induced

16. Sole release of NYPK from crosslinked rIL-1  when treated with Endo Lys C under native condition

17. Bioactivity measurement of various forms of crosslinked rIL-1  Crosslinked IL-1  , without the NYPK region is NOT active.

19. Crosslinked and EKC treated IL-1  is almost identical to native untreated IL-1 

20. Subtle conformational change detected between native and crosslinked IL-1  by fluorescence spec. Tryptophan Ex 295 Tryptophan & Tyrosine Ex 275 Crosslinked Native

21. Hydroxyl titration studies of tyrosine residues in native and crosslinked IL-1  Native Crosslinked