Molecular Weights of Hemoglobin and Myoglobin by Atomic Weight Absorption Spectrophotometry We have been mterested m addrne a molecular weleht exoenment to our schedule of exoerlmenta for our Instrumental
are required and the work can be easily completed in a two to three hour class period. We began by demonstrating the use of the atomic absorption instrument. Our lab assistants prepared a stock solution of hemoglobin containing 1694.8gg/ml. The students made 1:4 dilutions of the stock solution to obtain a working solution containing 423.7 figlml. A similar procedure is used for myoglobin. To facilitate dissolution of the two proteins we added two drops of concentrated hydrochloric acid and about 25 ml of water. The mixture was shaken gently for a l-hr period until no solid protein was visible. Both solutions were brought to the mark with distilled water and refrigerated until about one hour before they were to be used. Iron standards were prepared from a stock solution by dissolving a known mass of 0.009-in. Baker analyzed reagent iron wire in 6 M HCI. Five standards were prepared by the instructor, and their absorbance values were obtained as part of the demonstration. The students recorded this information and used it to determine a standard curve. We deliberately chose our highest concentration at the upper limit of the curve toshaw that thelinear relationship does break down a t higher concentrations. At this paint the students, working in pairs, may aspirate their protein solutions and determine the iron absorbance. A sample calculation for hemoglohin follows: &, ..= 0.100 lo-'
= 2.510 X 10-8 moles 55.8 glmole mass Hb in one ml = 423.7 fig
nF' =
1.40 figlml 2.510 x moles Fe= 6,275 nHb = 4 moles Felmole Hb 423'7 = 67,522 glmole Molecular Weight Hb = 6.275 X 10W mole Cpe =
moles
The eighth edition of the Merck Index gives 68,000 as the molecular weight of hemoglobin, but 65,000 is the currently accepted value. We estimate our experimental uncertainty as f5%. The myoglohin solution we used was deliberately prepared to give iron values near the upper limit of our standard curve. We found 2.86 fig iron for our myoglobin solution. The calculated molecular weight is 20,617 glmole. This value is well above the accepted value of 17,000 glmole and is outside our estimated experimental error. This discrepancy might be caused by the choice of concentration for the myoglobin solution, though the curve is apparently linear in this range. Another possibility is the myoglobin sample may have been impure. Our protein had been previously used and was not refrigerated. We believe this is a good experiment because it demonstrates a number of important physical and analytical principles and applies these principles to meet a need in the Biochemistry area. John D. Worley and Tom Londo St. Norbert College DePere. WI 54115
Journal of Chemical Education
