人血清白蛋白与3,5-二碘水杨酸相互作用的分析

人血清白蛋白（HSA）是血浆中含量最丰富的蛋白质。HSA与药物化合物和其他体内物质结合，并在这些物质向靶器官的运输中发挥重要作用。一些研究已经报道了HSA的结合亲和力及其与多种化合物的相互作用。本应用说明演示了使用ATS-530自动滴定仪和J-1500 CD光谱仪获得 3,5-diiodosalicylic滴定到HSA的CD光谱，以确定解离常数。Application NoteCDSSpectroscopy Analyzing the interaction of human serumalbumin and 3，5-diiodosalicylic acid2/3Application Note Analyzing the interaction of human serum albumin and 3，5-diiodosalicylic acid probed by circular dichroism Introduction Hu man se ru m a lbu mi n (HSA) is th e mos t abu n da n t pro t e i n in bl ood p l a sm a. HSA bin ds with p h ar m a c e u ti cal c o m p ou n ds an d o th e r in -v iv o subst a n c es a n d pl a y s a n imp o r tan t r o l e in the tra n spor t of th ese s u bs t a n ces t o t a r get or g ans. Seve r a l s t u d i es hav e r e p o rte d o n th e bind i n g a ffi n it y o f HSA a nd it s in t e r a c ti o n s w ith a v a riet y of c o mp o u n ds.3 JASCO J -1500 CD spectrometer Th is app li c a tion n ote s dem o n s tr at es th e u s e o f the J -1500CD spect r omete r a n d ATS-530 automatic titra t or to monito r th e in ter a cti o n of HSA tit rate d w i t h 3，5-d ii o do s ali cy li c a c i d.W hile 3，5-di i o d os a l i cyl i c a c i d i s a ch i ral， i ts i n t e ra cti on w ith chiral HSA i n du c es circ u la r d i c h roism . View product i nformation at w ww .jas co i n c.c OM Keywords J-1500， c ir c u l a r di c h r o i sm， HSA， p h a rma c eu ti c a l s ， ATS-530au t o mati c titra t o r ， bi oche m is try ， p h ar ma c e u tical Experimental Measurement conditions Data acquisition interval 2 sec Scan speed 100 nm/min Spectral bandwidth 1nm Data pitch 0.1nm Path length 10 mm Accumulations 2 28600 Mary's Court ， Ea s t on， MD 21601 U S A 50 mL al i q u ots of 0.025 mM of 3，5-diiodosali c ylic aci d wa s added t o 2 m L o f 0.0228 m M o f HSA i n 100 mM acetate buf f el (pH 6.3) 20 t ime s . Th e CD sp ectru m was s ca n ne d f r o m 360 t o 260 n m and also monit o r e d a t 320 n m. Results Chir a lity c an b e in duced in an ac h i r a l su b st a nce i n t e r actin g wi th a ch i ra l s u b s t a n ce. Thi s in t er a ctio n wi ll e xh ib i t c irc u l ar d i c h roism . The r esu lti ng CD spectr u m f ro m the in t e r action of ach ir a l 3，5-di i odosalicylic acid w ith c h iral H SA sho w S a p ositive pe ak a t 320 n m in Fi g ure 1. While H S A d oes not s h ow a CD s i g n a l a t 320 n m， the ind u ced CD from the in t e ra ction of HS A an d 3，5-d ii o d o s a licyli c a ci d d ep i ct s th i s si gna l at 320 nm w h i c h in cre a ses w i th th e in crea s in g ad d it io n s of 3，5-d iiodosali c ylic acid and is sho wn in F i g ure 2. Figure 1. 3-D graphical representation of the CD spectrum of HSA titrated with 3，5-diiodosalicylic acid. Figure 2. CD spectra of HSA titrated with 3，5-diiodosalicylic acid. The arrows represent the i ncreasing volume of 3，5-diiodosalicylic acid. The i n crease in the indu c ed CD signal at 320 n m w as plotted in F i gure 3 a s a f u n tion o f the 3，5-di i odos a licylic a cid co n cen tr ation . F i g ur e 3 is also k no wn a s a Hill pl ot wh ich describes th e coope r ati v i t y of the in t e ra c tion be t wee n HSA a n d 3，5-d i iodosa l icylic ac i d . A dissociation constan t ， w h i c h i s th e co n cen tr ation at w hi c h 50% of a s u btsa n ce is bou n d ， can a lso be est i m a ted f rom a Hill pl ot . Us in g Figu r e 3， the disso c i a tion co n sta nt of 3，5-diiodosalicylic acid binding to HSA was d et er m in e d to b e 0.023 m M . The Hil l c o effi c ie n t i s a ppr o x ima t el y 3.1 wh i c h i n dic a te s a po s itiv e coo per a t i v e r ea c tion. 28600 Mary's Cour t ， E asto n ， MD 21601US A Figure 3. CD signal monitored at 320 nm for the t i t ration of 3，5-diiodosalicylic acid to HSA. Conclusion Th is ap p lic a t i on no te demonstr a te s th e use of th e AT S -530 a u t om ati c t itra t o r an d J -1500 C D sp ectr o meter t o o b t ain CD s pe c t r a of t he t i t ra ti o n of 3，5-d ii o d o sa l i c y l i c t o HSA i n o rder t o dete r mine a d i ssoc i a ti o n co n s t a n t . References 1. R . Br oderse n， J . Bi ol . C h e m. (1977) 252， 14，5067-5072. 2. U . Kr ag h -Ha n se n ， Bi oc h e m . J . (1983) 209， 135-142. 3. Si n h a ， S. S.， Mit r a ， R. K.， and S. K. P al ， J . P h ys . C h em .B. (2008)112，4884-4891. 28600 Mary's Cour t ， E asto n ， MD 21601US A