Routine electrophoretic surveys of human blood samples have revealed hundreds of variant hemoglobins. Most are rare and differ from normal hemoglobin by a single amino acid substitution. Some like HbS (Sickle Cell Hemoglobin) produce severe clinical problems; others produce no ill effects. This problem set will deal with two different hemoglobin variants which affect the beta-chain. Hemoglobin-G Coushatta was reported in two groups of Native Americans and is harmless. Hemoglobin Southampton was discovered in England and causes severe hemolytic anemia. Each variant has been purified and its sequence determined. The 146 amino acid sequence for the normal human hemoglobin beta-chain is given below for reference in the questions that follow.

 1                                  10                                Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-Val-Thr-Ala-Leu-Trp-
 
                 20                                      30
 Gly-Lys-Val-Asn-Val-Asp-Glu-Val-Gly-Gly-Glu-Ala-Leu-Gly-Arg-

                                    40                               Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-Phe-Glu-Ser-Phe-

                 50                                      60
 Gly-Asp-Leu-Ser-Thr-Pro-Asp-Ala-Val-Met-Gly-Asn-Pro-Lys-Val-

                                    70                             Lys-Ala-His-Gly-Lys-Lys-Val-Leu-Gly-Ala-Phe-Ser-Asp-Gly-Leu-

                 80                                      90
 Ala-His-Leu-Asp-Asn-Leu-Lys-Gly-Thr-Phe-Ala-Thr-Leu-Ser-Glu-

                                    100                            Leu-His-Cys-Asn-Lys-Leu-His-Val-Asp-Pro-Glu-Asn-Phe-Arg-Leu-

                 110                                     120
 Leu-Gly-Asn-Val-Leu-Val-Cys-Val-Leu-Ala-His-His-Phe-Gly-Lys-

                                    130                            Glu-Phe-Thr-Pro-Pro-Val-Gln-Ala-Ala-Tyr-Gln-Lys-Val-Val-Ala-

                 140                     146
 Gly-Val-Ala-Asn-Ala-Leu-Ala-His-Lys-Tyr-His
 
 

        The purified hemoglobins were aminoethylated (a specific chemical modification of cysteine residues that makes them similar to another common amino acid) and subjected to trypsin hydrolysis. The resulting peptides were separated by a combination of electrophoresis and paper chromatography and compared to normal hemoglobin. In each case, only one peptide differed from the normal hemoglobin peptide map. That peptide was purified and hydrolyzed in HCl for amino acid determination.  The amino acid composition of the affected peptides is shown in the table below.
 

Source of Variant Peptide	Amino Acid Composition
	Arg	Asx	Glu	Pro	Gly	Ala	Val	Leu	Cys
Hemoglobin G-Coushatta	1	2	1	0	3	2	3	1	0
Hemoglobin Southampton	0	1	0	1	1	0	2	2	1

 

1. From the above compositions and knowledge of the procedures used to obtain the peptides, deduce the location of each peptide in the overall sequence.  Describe how you arrived at your answer.   How sure are you of your assignment?

2 . What is the amino acid substitution that has taken place?  If possible, give the position number and the amino acid that normally occurs at that position.   Would the substitution affect the electrophoretic mobility of the intact hemoglobin molecule?

3. Based on your understanding of the structure of hemoglobin and the particular properties of the amino acid, propose a consistent hypothesis which explains the severe hemolytic anemia for patients with Hb Southampton and the benign effects of hemoglobin Coushatta. (Hint: Locate the substitution within the overall three-dimensional structure of hemoglobin.)

4. For future reference in studying for the third hourly exam and the final, can you deduce the nature of the base replacement mutation that occurred in each case?  (Figure it out, before you check your answer.)