<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; ">Hi Jack and MItra,<div> One of the ways to minimize damage from excess dithionite is to use a procedure described by Dixon and McIntosh, Nature (London) 213, 399 (1967). Essentially one uses a G25 Sephadex (or equivalent )column to load a band of dithionite and then follow with your heme protein. The protein obviously migrates faster through the column and the time in contact with dithionite is greatly reduced. I tried this once long time ago with myoglobin and showed identical absorption spectra and oxygen equilibrium measurement with oxy myoglobin prepared directly from whale tissue (those were the good old days in Frank Gurd's lab at Indiana). At any rate check out the reference and give that a try. </div><div> Steve<br><div><div>On Feb 5, 2009, at 8:03 AM, Jack Kornblatt wrote:</div><br class="Apple-interchange-newline"><blockquote type="cite"><div>Hello Mitra<br>Chance, I think, once described dithionite as man's worst enemy. the concentration that you are using is far in excess of what is need to keep your solutions anaerobic. The reaction products of dithionite are too numerous to list even if I could remember them. If you degas your solutions just before loading and then add dithionite to 1 mM this should give you the desired "low" oxygen.<br>Is it really necessary to keep dithionite as low as possible? I have little experience with Hb but if we use dithionite and cytochrome c oxidase at 11 mM there are sufficient biproducts generated that interpreting data is very difficult<br><br>best<br>jack kornblatt<br>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@rasmb.bbri.org">RASMB@rasmb.bbri.org</a><br>http://rasmb.bbri.org/mailman/listinfo/rasmb<br></div></blockquote></div><br><div apple-content-edited="true"> <span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><div style="word-wrap: break-word; -khtml-nbsp-mode: space; -khtml-line-break: after-white-space; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Geneva" size="3" style="font: 12.0px Geneva; font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; "><span class="Apple-style-span" style="font-family: Geneva; ">Steven J. 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