Table 1.

Elution of Proteins from Heparin and SP-Sepharose Resin^a

protein	Hep [NaCl] (mM)	Δ[NaCl]H (mM)	S-Seph [NaCl] (mM)	Δ[NaCl]S (mM)	ΔΔ[NaCl] (mM)
W55D	920	–	590	–	–
K8A	820	100	540	50	50
R9A	680	240	610	−20	260
R18A	620	300	450	140	160
R23A	650	270	460	130	140
K25A	820	100	850	−260	360
R35A	640	280	490	100	180
K42A	570	350	420	170	180
R43A	550	370	420	170	200
K46A	810	110	470	120	−10
R61A	770	150	520	70	80
R70A	760	160	530	60	100
R23A/R43A	410	510	330	260	250
W55D 1–72	920	0	660	−70	70
CC0b	460	460	410	180	280
CC3	440	480	470	120	360
WT	760	160	460	130	30

^aProtein elution was monitored as a function of NaCl concentration (Hep [NaCl] and S-Seph [NaCl]). Δ[NaCl]^H and Δ[NaCl]^S values were calculated by subtracting heparin and SP-Sepharose elution from W55D. The value of Δ[NaCl]^S was then subtracted from Δ[NaCl]^H to remove the electrostatic contribution from the protein–heparin interaction: ΔΔ[NaCl] = Δ[NaCl]^H − Δ[NaCl]^S. ΔΔ[NaCl] refers to the heparin specificity index. The larger the ΔΔ[NaCl] values, the greater the effect of the mutation on specific heparin binding. All variants with the exception of the following were made in the XCL1 W55D background: CC0, CC3, and WT.

^bCC0 is an unfolded variant of XCL1. Mutation of C11 and C48 to alanine removes the disulfide bond.