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Determination of haptoglobin types by electrophoresis in acrylamide gel
792
SHORT
Determination
COMMUNICATIONS
of haptoglobin types by
electrophoresis in acrylamide gel* The serum haptoglobins (hemoglobin-binding proteins) migrate electrophoretically on paper as a,-globulins, but when separated by starch or acrylamide gel electrophoresis they separate into several fracticns which prcduce patterns of 3 general types. The buffer, Tris-veronal, PH 8.4 is prepared by dissolving 3.6 g scdium barbital, 3.1 g barbituric acid, and 2.9 g Tris (hydroxymethyl) aminomethane in distilled water and making up to I 1 with distilled water. A 5% solution of Cyanogum 41 ** is prepared by dissolving IO g Cyanogum 41 in 150 ml of buffer. Filter and make up to 200 ml with the buffer. Carefully pipet 1.0 * * into the gel solution and mix gently ml of DMAPN (/--dimethylaminopropionitrile) by rotation. Add 0.5 g of ammonium persulfate and mix carefully as before. Immediately pour the catalyzedgel solution into the vertical cell***, avoiding the Insert the sample slot former and allow the gel to form. formation of bubblesI. Gelling is usually started within 3 min and is completed in 15 min2. A complex of haptoglobin and hemoglobin is prepared by adding 20 ,~l (0.020 ml) of fresh 10% normal adult hemoglobin solution to 1.0 ml of fresh, clear, nonhemolysed serum. Carefully pipet 25 ,~l (0.025 ml) of this hemoglobin complexed serum into the preformed sample slots (1.5 cm long). Allow the specimens to settle in the sample slots for 2-3 minutes and apply a constant voltage of zoo V for 4 h. After electrophoresis the gel is removed from the cell and stained with a benzidine stain which is prepared by heating 200 ml of distilled water and 1.0 ml of glacial acetic acid to just below boiling. Remove from heat and carefully add 0.4 g of 3,3’dimethoxybenzidine*** *. Mix by rotation. Allowtocool to room temperature and filter. Just prior to staining, add 0.2 ml of 30% hydrogen peroxide and mix by rotation. The gel should be completely submerged in the stain. After 45-60 min, depending upon the thickness of the gel, the gel is washed 3 times in rj”/o acetic acid solution. To facilitate removal of precipitated stain from the surface of the gel, wipe the gel gently with a soft sponge or cotton while the gel is submerged in the acetic acid wash solution. In order to locate the haptoglobin fractions with respect to other serum protein fractions a second electrophoretic separation was carried out using the same serum specimens and procedure as before. After electrophoresis the second gel was stained with Amido-black stain and the background stain removed electrically3. Fig. I illustrates the 3 common haptoglobin types according to the nomenclature of SMITHIES~. It will be noted that the haptoglobin fraction with the fastest mobility corresponds to the fraction labelled PC (transferrin). This transferrin component was identified by tagging the serum with 59Fe.
* The opinions cxprcssed herein are those of the authors and cannot be construed as rcflecting the views of the ?Tavy Department or of the Naval Service at large. Usage of commercially available materials cannot be construed as implying endorsement of those products over other similar products. ** American Cyanamid Co., New York, N.Y. *** E-C Apparatus Co., Philadelphia, Pa. ** * * Distillation Products, Eastman Kodak Co., Rochester, N.Y. Clin. Chim. Actn, 8 (1963)
7gz-793
793
SHORTCOMMUNICATIONS
%(B) _.
(A)
(8)
(C)
tip 2-l
Hp f-t
Fig. I. Vertical gel electrophoretic patterns of human serum in acrplamide gel showing the 3 typical haptoglobin types. (A), HpHgb complex. Benzidine stain; (R), Hp-Hgb complex. Amido-black stain; (C), Serum. Amido-black stain.
An examination of the 3 haptoglobin types illustrated in Fig. I will reveal that the haptoglobin type I-I is readily distinguishable from types Z-I and z-z in the uncomplexed serum patterns stained with Amido-black. However, the difference between types Z-I and z-z (uncomplexed serum) is not readily apparent and it is necessary to complex the haptoglobins with hemoglobin, separate by electrophoresis, and stain with benzidine in order to differentiate these haptoglobin types. T. G. FERRIS R. E. EASTERLINC R. E. BUDD
Rear Admiral Calver’s Physical Chemistry Research Laboratory, Room 322, U.S. Naval Medical School, NNMC, Bethesda, Md. (U.S.A.)
1 T.G. FERRIS, R. E. EASTERLING AND R. E. BUDD, Blood, rg (1962) 479. 2 T. G. FERRIS, R. E. EASTERLING AND R. E. BUDD, Am. J. Med. TechnoZ.,(1963) 3 T. G. FERRIS, R.E. EASTERLING AND R. E. BUDD, Am. J.Clin.Pathol.,39(rg63) * 0. SMITHIES, Biochem. J., 61 (19553 629.
Received
February
in the press. 193.
8th, 1963 Clin. Chim.
Acta, 8 (1963)
792-793
SHORT
Determination
COMMUNICATIONS
of haptoglobin types by
electrophoresis in acrylamide gel* The serum haptoglobins (hemoglobin-binding proteins) migrate electrophoretically on paper as a,-globulins, but when separated by starch or acrylamide gel electrophoresis they separate into several fracticns which prcduce patterns of 3 general types. The buffer, Tris-veronal, PH 8.4 is prepared by dissolving 3.6 g scdium barbital, 3.1 g barbituric acid, and 2.9 g Tris (hydroxymethyl) aminomethane in distilled water and making up to I 1 with distilled water. A 5% solution of Cyanogum 41 ** is prepared by dissolving IO g Cyanogum 41 in 150 ml of buffer. Filter and make up to 200 ml with the buffer. Carefully pipet 1.0 * * into the gel solution and mix gently ml of DMAPN (/--dimethylaminopropionitrile) by rotation. Add 0.5 g of ammonium persulfate and mix carefully as before. Immediately pour the catalyzedgel solution into the vertical cell***, avoiding the Insert the sample slot former and allow the gel to form. formation of bubblesI. Gelling is usually started within 3 min and is completed in 15 min2. A complex of haptoglobin and hemoglobin is prepared by adding 20 ,~l (0.020 ml) of fresh 10% normal adult hemoglobin solution to 1.0 ml of fresh, clear, nonhemolysed serum. Carefully pipet 25 ,~l (0.025 ml) of this hemoglobin complexed serum into the preformed sample slots (1.5 cm long). Allow the specimens to settle in the sample slots for 2-3 minutes and apply a constant voltage of zoo V for 4 h. After electrophoresis the gel is removed from the cell and stained with a benzidine stain which is prepared by heating 200 ml of distilled water and 1.0 ml of glacial acetic acid to just below boiling. Remove from heat and carefully add 0.4 g of 3,3’dimethoxybenzidine*** *. Mix by rotation. Allowtocool to room temperature and filter. Just prior to staining, add 0.2 ml of 30% hydrogen peroxide and mix by rotation. The gel should be completely submerged in the stain. After 45-60 min, depending upon the thickness of the gel, the gel is washed 3 times in rj”/o acetic acid solution. To facilitate removal of precipitated stain from the surface of the gel, wipe the gel gently with a soft sponge or cotton while the gel is submerged in the acetic acid wash solution. In order to locate the haptoglobin fractions with respect to other serum protein fractions a second electrophoretic separation was carried out using the same serum specimens and procedure as before. After electrophoresis the second gel was stained with Amido-black stain and the background stain removed electrically3. Fig. I illustrates the 3 common haptoglobin types according to the nomenclature of SMITHIES~. It will be noted that the haptoglobin fraction with the fastest mobility corresponds to the fraction labelled PC (transferrin). This transferrin component was identified by tagging the serum with 59Fe.
* The opinions cxprcssed herein are those of the authors and cannot be construed as rcflecting the views of the ?Tavy Department or of the Naval Service at large. Usage of commercially available materials cannot be construed as implying endorsement of those products over other similar products. ** American Cyanamid Co., New York, N.Y. *** E-C Apparatus Co., Philadelphia, Pa. ** * * Distillation Products, Eastman Kodak Co., Rochester, N.Y. Clin. Chim. Actn, 8 (1963)
7gz-793
793
SHORTCOMMUNICATIONS
%(B) _.
(A)
(8)
(C)
tip 2-l
Hp f-t
Fig. I. Vertical gel electrophoretic patterns of human serum in acrplamide gel showing the 3 typical haptoglobin types. (A), HpHgb complex. Benzidine stain; (R), Hp-Hgb complex. Amido-black stain; (C), Serum. Amido-black stain.
An examination of the 3 haptoglobin types illustrated in Fig. I will reveal that the haptoglobin type I-I is readily distinguishable from types Z-I and z-z in the uncomplexed serum patterns stained with Amido-black. However, the difference between types Z-I and z-z (uncomplexed serum) is not readily apparent and it is necessary to complex the haptoglobins with hemoglobin, separate by electrophoresis, and stain with benzidine in order to differentiate these haptoglobin types. T. G. FERRIS R. E. EASTERLINC R. E. BUDD
Rear Admiral Calver’s Physical Chemistry Research Laboratory, Room 322, U.S. Naval Medical School, NNMC, Bethesda, Md. (U.S.A.)
1 T.G. FERRIS, R. E. EASTERLING AND R. E. BUDD, Blood, rg (1962) 479. 2 T. G. FERRIS, R. E. EASTERLING AND R. E. BUDD, Am. J. Med. TechnoZ.,(1963) 3 T. G. FERRIS, R.E. EASTERLING AND R. E. BUDD, Am. J.Clin.Pathol.,39(rg63) * 0. SMITHIES, Biochem. J., 61 (19553 629.
Received
February
in the press. 193.
8th, 1963 Clin. Chim.
Acta, 8 (1963)
792-793