Determination of the specific heat capacity of hemoglobin- and methemoglobin-water mixtures using an adiabatic calorimeter

The specifk heatcapacityof bovine hemoglobin, ~&etbemogIobin-;and thermaiiy denaturedhem&obiwwater mixtwes were meaknxl iti the tempemturerange from 10 to 80°C. The partialspecificbeat capzities for mass fractionsof the protein between0 and 1 werecomputed. Signiikmt diffkrencesof the partialquantitieswere obtainedfor the native,respxtivdy denat&i stateof protein and f6r the proteinin the native s&Uein fiuid-mktuns, rcspectivtiyin ratherdry mktures, Formixtu~~ with proteinmass fracrionsup to 0.45, exkeedingthe value in living human red cells, partial specik heat capacitiesof cithercornpone~~~ts are found to ix constant The aoauacy of the used adiabaticcalbrimeterwill be describedbriefly- I I INlRODKHX’iO~

a preciseadiabaticmicn5-caIorimeter has been develoH1-4 for measuringcontinuousIythe specik heat opacity of small liquid or solid Samples in the temperatureran& from 10 m 80°C subject-toan instrUmentat uncerknty of less than 6mJ KT' -or about O-3%.for-a typicai sample mass of 0.6g wa& The precisionandaccuracya&ieved are requiredin o&&r ti &a+e partialsptdfic heat capacitiesofthecomponentsinamixtunzofwakwithptiteikandothercom$onents of biologicalinterestThe valuesof the partialquantitiesshouldgivesome information about the interactionsof pro&& with water,especia!lyat high proteinconc&tr%tions -inthe rangeof 20 to 40 wt; % rcscmblingthe massfractionof proteinsin thccytopksm of livkg cell From-the partialspe@c beat _ety of +ater in ticb simpk model syw .we hope to obtain-iaformko~: about &e state of k&z in living cell& ‘_ -c--.- -_Micr~o~ for the s+n& pur&e but of some&at di&rent design&x-e &&iby-~&‘-~d byS&&&&W&s__ ‘I _. - __ : . ._ :

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ThecaIorimeter-definedastbatpartof theapparatusin~hichheatisaaxmteIy accounted for-is a copper tribe of IO mm outer diame&rand of 50 mm Iength. In the caIorimeter a small glass ampoule with the sample was pIaczd:The sampk mass ranged between O-3 and 1 g The temperature dif&ren~ between the calorimeter and the adi&atic jacket was measured by an 8-junction thermopile- The adiabatic jacket temperatuti‘waS measured by a quartz thermometer 2801 A of Hewhztt-Packard using-a HewiettPackard quariz probe 2850 k The resolution of the digitaI thermometer amour&d to 1 - 1o-5 K with a sampie time of 100 s. Due to the presence of a second and third temperature~ontjackef it was possibIe to keep the amplitude ofthetemperature oscillations between calorimeter and adiabatic jacket below 3 - 10m5K. The diEerenee between time and average temperature was smaIIer because of the rather symmetric temperature oseihations; The relative accuracy. when tin - - g a~ tempemture intenaI of typical 0.2 K in 100 s, corresponding to a rate of temperature increase of 8 K Per hour, was about 5 - 10V4. _ The potential difference at the heater and the heater current was measured by a classical potentiometer assembly of hig6 precision instruments obtained from Leeds and Northrup. The reIative determination accmacy of the ekctric power in the calorimeter heater was better than 2 - lo-*_ The reIative overah determination accuracy of the spa%% heat due & the determination uncertainty of ekctric power and temperature increase with time was better than 1 - 10m3_The corrections of systematic errors introduced an uncertainty of the same order of magnitude- The experimentahy d _ _ ed overall a&xuacy of about 0.3% with a typicaI liquid sampIe of about 0.6 g is based on the known sma.Il irreproducibility of the charging and discharging procedure_ AII errors are consenrativeIy stated_ Details wiII be described in ref, 4, In order to demonstrate the reproducibility and the a&racy of the calorimeter, two measurements from the specific heat capacity of about 0.6 g warer samples in the temperature range between about 17 and 80°C are shown The values deviate from the standardvaIuesgbyaboutO~I%(Fig, 1) .

Bovine oxyhemogIobin (BHb) was prepared from fi-esh bIc+and purified as described in ref. IO_ Boviie methemogiobin (BMetHb) was prepared by IyophiIiaing BHb. Sampks with di&ent protein JEW fraction w were Ii&d into thegtassampouks mentioned above znd Introduced into the calorimeter_ The protein mass fiactioe\- as determinerI by dry mass determin;rton with a &at&e uncerminty of a few pcT”hijiFigure 2 shows the computer plot of a calorimeter scanof a,lT.!i wt. %;w wa$r mixture. In the tempemture range of +$xestf between 10 and *oC_:the.tL . I Gti f& & pbin& -$,& -fiti .by &.fk&jn cp = & ;--i’i C-T + &_’ T-2,

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