Figure 1.
HPLC elution profile of the purified fraction of GpA72 Sera from four to six arthritic rats were pooled and subjected to protein purification as described under the methods section.
The single peak eluting at 21.225 min represented GpA72.
Figure 2.
The protein was hydrolyzed with 6N HCl under vacuum at 110°C for 24 h. Amino acid analysis was performed by pre-column derivatization with phenylisothiocynate. The phenylthiocarbomoylamino acids were analyzed using a Pico Tag column (3.9×150 mm)on a Waters HPLC system, equipped with a 1525 binary pump and Waters 2996-photodiode-array (PDA) detector set at 254 nm. Numbers on the peaks are retention times in minutes.
Table 1.
Comparison of the amino acid compositions of GpA72 and α-Cysteine protease inhibitor.
Figure 3.
Identification of GpA72 as T-Kininogen I by tandem mass spectrometry analysis.
Protein band corresponding to GpA72 was excised and protolysed with trypsin. Extracted peptides were analyzed by liquid chromatography Tandem mass spectrometry using an ion trap mass spectrometer (LCQ Deca XP, Finnigan) as described in the Methods section. Top panel shows CID spectrum that was matched to 66DGAETLYSFK75 of T-kininogen 1. Observed b- and y-ions are indicated. Whole protein sequence and the peptides identified by LC-Tandem MS (bold) are shown in the bottom panel. Peptide sequences identified that aid in distinguishing the T-Kininogen I from T-Kininogen II are underlined.
Figure 4.
Serum levels of T-kininogen I in rats injected with different proinflammatory agents.
Representative native PAGE gel image of sera from rats with hind joint injections of the following: Lane 1: Liquid paraffin oil, Lane 2: Adjuvant containing H37Rv, Lane 3: Turpentine oil; Lane 4: Zymosan; Lane 5: Carrageenan and Lane 6: Collagen.
Figure 5.
Lowering of T-Kininogen I levels by dietary curcumin and capsaicin.
Representative native PAGE gel image of sera from rats with hind joint injections as follows: Lane 1: Liquid Paraffin oil, Lanes 2–8: Adjuvant containing H37Rv. Lanes 3–8 are serum samples from rats orally administered with various compounds as follows: Lanes 3 and 4: Curcumin; Lanes 5 and 6: Piroxicam, Lanes 7 and 8: Capsaicin.