Methods used for protein cleavage are all adapted from methods outlined in AlIen, ( 1 98 1 ), Smith, ( 1 994a) and Smith, ( 1994b).
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2.2. 1 1 . 1 Cyanogen Bro mide (Methionine-X)
Pure protein was dissolved in water to a final concentration of 1 -5 mglml. One volume of ammonium bicarbonate (0.4 M, pH 8.4), and 1 -5 % (v/v) 13-mercaptoethanol were added. After passing nitrogen gas over the solution, the solution was incubated at room temperature for 1 8 hours. The solution was then dried down under vacuum, and redissolved in formic acid to 1 -5 mglml. Water was added to bring the final concentration of formic acid to 70 % (v/v), and excess crystalline cyanogen bromide added ( 1 -2 crystals). This was incubated at room temperature for 24 hours, then dried down under vacuum, or lyophilised.
2.2. 1 1.2 Dilute Acid Cleavage (Aspartate-X)
The protein was dissolved to 1-2 mglml in 30 mM HCl in a glass hydrolysis tube. The hydrolysis tube was sealed under vacuum and incubated at 1 08°C for 2 hours. The tube was then opened, the sample diluted with water, and the solution lyophilised.
2.2. 1 1.3 Trypsin (Lysine-X and Arginine-X)
The protein was diluted to -2 mglml in water. bicarbonate solution (0.4 M pH 8 .4) was added.
An equal volume of ammonium A number of tests showed good cleavage to occur after a 5 minute incubation at room temperature. Digestion was terminated by adding 1 mM PMSF.
2.2. 1 1.4 Thermolysin (X-Hydrophobic)
To 20 �l of -2-5 mglml protein was added 5 �l of ammonium bicarbonate solution (0.4 M pH 8.4), 1 1 �l H20, and 4 �l 1 mM CaCh. A small amount of solid thermolysin was added and the solution incubated at 45°C for 1 hour. 1 mM EDT A was added to
stop the reaction, and the solution was then dried down under vacuum.
2.2. 1 1.5 Leucine Aminopeptidase (N-terminal A mino Acids)
To 20 �l of 2-5 mglml protein was added 80 �l 0. 1 M Tris.CI containing 2.5 mM MgCh. The solution was incubated at 3 7°C for 1 0 minutes, frozen for 3 0 minutes at -70°C, and dried down under vacuum.
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2.2. 1 1.6 Separation of Pep tides Generated by Cleavage
Two methods were commonly used to separate peptides generated by cleavage: tricine gels, and HPLC based separation.
3-layer tricine gels were constructed as outlined in section 2. 1 . 1 1 , from the method of Schagger & von Jagow, ( 1987). After running the peptide mixture through the 3 -layer gel, the gel was electroblotted onto PVDF membrane for 2 hours at 2 5 0 rnA in 1 0 mM
CAP S . The PVDF membrane was then briefly stained with Coomassie blue solution, and destained in methanol until bands could be seen. Bands of interest were cut out, and frozen at -20°C until sequencing could be carried out.
To separate mixtures of peptides by HPLC, the mixture was applied to a C 1 8 column pre-equilibrated in 0. 1 % TF A in water. A gradient was then run over 20-30 minutes to
1 00 % acetonitrile, separated peptides were trapped as they eluted, and sequenced.
2.2 . 1 2
P rotein Sequencing
Pure proteins and peptides generated by protein cleavage were sequenced on an Applied Biosystems 476A Automated Protein Sequencer, using sequential Edman degradation. Sequencing was performed by Ms. Jo Mudford
2.2. 1 3
Amino Acid Analysis of P roteins
The amino acid composition of pure protein was determined by total acid hydrolysis in 6 M HCl under vacuum, and then applying the amino acid mixture to a Pharmacia LKB Alpha Plus Amino Acid Analyser. Amino acid analysis was carried out by Ms. Debbie Frumau.
2.2. 1 4
N-Terminal Deacylation
The method used to deacylate purified protein with a blocked N-terminus was that described by Hirano et ai., ( 1 992). E ssentially, this method involves cleaving the purified protein and blocking the generated internal N-termini with phenylisothiocyanate. The original N-terminal residue is then removed with the enzyme N-acylaminoacyl peptide hydrolase, and the mixture applied to the automated sequencer as usual. The
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only free N-terminus should be the actual N-terminus. The cleavage is necessary as the deacylating enzyme does not work efficiently on peptides longer than 1 0-20 residues.
Pure protein was run on an SDS gel, and electroblotted onto PVDF membrane as
previously described. The stained band was cut out and treated with 0 . 5 % (w/v) polyvinylpyrrolidone in 1 00 mM acetic acid at 3 7°e for 3 0 minutes to block the unbound protein-binding site. The membrane was then washed with water 1 0 times, and digested with trypsin in 0 . 1 M ammonium bicarbonate pH 8 containing 1 0 % (v/v) acetonitrile at 3 7°e for 24 hours with shaking. The digestion buffer containing tryptic peptides was then transferred to an microfuge tube, and the membrane washed by vortexing with 1 00 III H20 and combined with the peptides. The buffer was evaporated to dryness, and 1 00 III of 5 0 % (v/v) pyridine and 1 0 III phenylisothiocyanate were added to bind with free but not blocked N-terminal amino acids of the tryptic peptides. The mixture was purged with nitrogen gas for 20 seconds, and incubated for 1 hour at 60°C. One ml of benzene/ethyl acetate ( l : 1 (v/v» , was added, and the solution vortexed and centrifuged at 3 000 x g for 1 minute. The supernatant containing reaction by-products and excess reagents was discarded, and this washing procedure was repeated three times. The sample was then evaporated to dryness under vacuum. Pre-made performic acid ( 1 00
ml)
was then added to the dried sample and kept at ooe for 1 hour, then evaporated to dryness under vacuum. Filtered water ( 1 00 Ill) was added and the drying was repeated. A solution of the peptides in 1 00 III of 0 . 2 M phosphate buffer pH 7.2 containing 1 mM DTT and one unit of N-acylaminoacyl-peptide hydrolase dissolved in 5 0 III of the same buffer was then incubated at 37°e for 1 2 hours to remove the W -acetylated amino acid. The sample was then sequenced as usual. The only peptide with a free N-terminaI available for sequencing is the N-terminal peptide.2.2. 1 5
Western Blotting
The protein sample was run on an SDS gel as described. The proteins were then transferred to nitrocellulose membrane by electrophoretic transfer as described previously. After transfer, the filter was placed in a suitable container and incubated in 2 % B S A in PBS for 60 minutes at room temperature with shaking. The primary (anti AlDH 1 ) antibody was then appropriately diluted in 1 % BSA in PBS, and the filter incubated in this solution overnight at room temperature . The filter was washed for 3 x 20 minutes in 0.05 % Tween in PBS, and then incubated with appropriately diluted
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secondary antibody in 1 % B S A in PBS for 2 hours at room temperature with shaking. The filter was washed as before, and then the substrate reaction was performed as detailed previously either with ECL reagents, or 4-chloro-l -naphthol. When ECL reagents were used, bands were visualised by exposure to X-ray film.