Bioc~imica et Biophvsica Acta 874 (1986) 19 22 19 Elsevier BBA32634 Effects of hydration in the oxygenation and autoxidation of carbonmonoxyhemoglobin powder Rosemary Sanches, Washington L.B. Melo and Marcio F. Colombo lnstituto de F{sica e Quhnica de S~o Carlos - USP, S~o Carlos, SP 13560 (Bra:il) (Received 22 October 1985) (Revised manuscript received 29 July 1986) Key words: Hydration; Hemoglobin; Oxygenation: Autoxidation The oxygenation and autoxidation of lyophilized human carbonmonoxyhemogiobin (Hb-CO) kept in environments with different relative humidities were followed with time using visible absorption spec- troscopy. The sample kept at 68% relative humidity was the one with the greatest formation of methemoglo- bin (Met-Hb), while for increasing or decreasing hydrations the Met-Hb content decreased gradually. Besides the presence of Met-Hb, it was observed that the samples kept above about 68% relative humidity were converted to oxyhemoglobin (Hb-O2) , the samples kept in the range 45-68% relative humidity were a mixture of Hb-CO and Hb-O2, while the samples kept below about 45% relative humidity and the solution sample continued as Hb-CO. Hemoglobin has, therefore, two critical hydration values, 45% and 68% relative humidity, which correspond to about 0.12 and 0.18 g H 2 0 / g protein, respectively. Although several proteins have had their struc- ture revealed by X-ray diffraction and spectro- scopic techniques [1,2], very little is known about the structure-function relationship of most of them. One of these exceptions is hemoglobin (Hb), for which details of the structural changes that occur in the oxygenation-deoxygenation process are known [3,4]. Hemoglobin can have its activity checked by absorption spectroscopy, since the several Hb derivatives have characteristic absorp- tion bands in the 400-700 nm region [5]. The technique also allows one to identify Met-Hb, which is considered to be the first intermediate in the protein denaturation process [6]. As the pro- tein ages, this derivative is formed naturally in an autoxidation process [5]. The hydration of proteins has been studied by several techniques (Ref. 7 and references therein). Correspondence address: Dr. R. Sanches, Instituto de Fisica e Quimica de S~o Carlos - USP, Sgo Carlos, SP 13560, Brazil. In general, small structural changes are observed upon dehydration, which are reversed to the na- tive conformation if the protein reabsorbs water. Dehydration of deoxy-Hb and Hb-O 2 by lyophili- zation, however, produces Met-Hb, which is an irreversible modification [8]. Lyophilization of several Met-Hb derivatives (aquo, fluoride, cyanide and azide), on the other hand, shows totally re- versible conformational changes [9], but these de- rivatives are not functional. Infrared spectroscopy studies on the hydration of Hb-CO films also showed reversibility of the induced conforma- tional changes [10], and this is a suitable species for function studies. In this paper we report how the oxygenation and the autoxidation processes are affected by the dehydration of Hb-CO. From absorption spec- troscopy studies we estimate how much water is necessary for the occurrence of these processes. Hemoglobin was prepared from human blood following a standard procedure [11]. The conver- 0167-4838/86/$03.50 :~' 1986 Elsevier Science Publishers B.V. (Biomedical Division)