Table 1.

Summary of Tyr- and Trp-containing-peptides identified in the chymotryptic maps of native and nitroxide-modified Mb.

Peaka	Elution Time (min)	Peptideb	Amino acid sequence	Trp and Tyrc	Observedd (m/z)	Calculatedd (m/z)	Change in concentratione (%)
1	28.61	Y10	Arg139-Tyr146	Tyr146	950.44 (+1)	950.51 (+1)	(+)1.62 ± 0.05
2	32.94	Y2*	Asn12-Trp14	Trp14	418.18 (+1)	418.20 (+1)	(+)2.8 ± 1.4
3	33.48	Y6*	Lys96-Tyr103	Tyr103	513.70 (+2)	513.83 (+2)	(−) 3.2 ± 5.4f
4	33.79	Y6*	His97-Tyr103	Tyr103	449.75 (+2)	449.78 (+2)	(−) 3.2 ± 5.4f
5	34.84	Y6*	Lys98-Tyr103	Tyr103	381.17 (+2)	381.25 (+2)	(−) 3.2 ± 5.4f
6	37.09	Y1	Gly1-Trp7	Trp7	763.30 (+1)	763.32 (+1)	(−) 48.8 ± 0.1
7	37.76	Y1**	Gly1-Trp7	Trp7	762.27 (+1)	763.32 (+1)	(+) 46.8 ± 0.1g

^aPeak numbers are assigned in the order of retention time as shown in Fig. 3.

^bPeptides are labeled Y_x according to the expected chymotrypsin cleavage sites (C-terminal to F, Y and W residues); *, non-specific site of cleavage at the N-terminus; **, peptide Y₁ modified at the N-terminus.

^cTrp and Tyr residues present in the chymotryptic peptides.

^dObserved and calculated m/z values: (+1), +1 ions; (+2), +2 ions.

^eChanges in concentrations are for nitroxide-modified Mb relative to untreated samples as estimated from the HPLC peak areas at 280 nm. Seven peaks eluting between 40 and 60 min and absorbing at 214 nm were chosen as the internal standards. Values are mean ± S.D. (n = 3).

^fChanges in concentrations are for all Tyr-103-containing peptides (e.g. sum of peaks 3, 4 and 5) assuming similar extinction coefficients.

^gThe % gain of peak 7 was calculated relative to that of peak 6 in untreated samples assuming similar extinction coefficients.