Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes
Driss Mountassif1*, Tarik Baibai2*, Latifa Fourrat2, Adnane Moutaouakkil3, Abdelghani Iddar3, M’Hammed Saı¨d El Kebbaj1, and Abdelaziz Soukri2
1Laboratoire de Biochimie et Biologie Mole´culaire, Universite´ Hassan II-Aı¨n Chock, Faculte´ des Sciences Aı¨n Chock, km 8 route d’El Jadida BP. 5366, Maˆarif, Casablanca, Morocco
2Laboratoire de Physiologie et Ge´ne´tique Mole´culaire, Universite´ Hassan II – Aı¨n Chock, Faculte´ des Sciences Aı¨n Chock, km 8 route d’El Jadida BP. 5366, Maˆarif, Casablanca, Morocco
3Unite´ de Radio-Immuno-Analyse, De´partement des Applications aux Sciences du Vivant, Centre National de l’Energie, des Sciences et des Techniques Nucle´aires, BP 1382 RP, 10001 Rabat, Morocco
*Corresponding address. Tel:þ212-22-230680/84; Fax:þ212-22-230674; E-mail: baiman21@yahoo.fr (T.B.) &
drissmountassif@yahoo.fr (D.M.)
A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from human erythrocytes. The comparison between this rapid method (one step) and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of 150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD1).
Western blot analysis using purified monospecific poly- clonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 4388888C and 8.5, respectively. The kinetic par- ameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD1 were 20.7 and 17.8mM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.
Keywords glyceraldehyde-3-phosphate
dehydrogenase; immunoaffinity purification; human erythrocytes; characterization
Received: November 18, 2008 Accepted: January 5, 2009
Introduction
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12) is a highly conserved protein with a key role in the glycolytic pathway [1]. GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (G3P) in the presence of NADþ and inorganic phos- phate. GAPDH is well conserved during evolution, being a protein with native molecular weight in the range of 140 – 150 kDa and composed of four identical subunits of35 – 37 kDa [2].
The ubiquity and evolutionary conservation of GAPDH indicate a highly important physiological func- tion. Indeed, GAPDH intervenes in several processes; its binding to tubulin may affect microtubule structure and function [3]; its interaction with 5V and 3VUTR mRNA sequence binding may be related to translational regu- lation [4]; and also its binding to PKCL/E in secretory vesicles affects the cell transport properties [5]. Unique nuclear GAPDH associations involve Oct-1 transcription factor [6] and its interaction with promyelocytic leukemia nuclear bodies [4] that are located in transcriptionally active regions [7].
The presence of GAPDH is required in the nuclear multienzyme complex that recognizes DNA incorporated structural analogs [8] and its binding to telomeres regu- lates their structure [9]. Nuclear GAPDH binding to tRNA precursors is required for their intranuclear trans- port [10]. The role of GAPDH as an Ap4a binding protein [11] is indicative of its role in cell proliferation [12]. Its location in the nuclear envelope involves
the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmp026.
Advance Access Publication 15 April 2009
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GAPDH in nuclear membrane fusion [13]. The role of GAPDH in apoptosis [14] can be related to its DNA binding properties [15].
The traditional procedure for preparing the GAPDH has some serious disadvantages. The procedure requires long periods of waiting at different steps especially during dialysis and the quality of the obtained enzyme is not reproducible. Since new resins and methods are available nowadays, the purification of glyceraldehyde-3- dehydrogenase from human erythrocytes has been reexa- mined and a new method for rapid purification has been devised. The objective of this study is to establish a good protocol that allows us to have a significant quan- tity of proteins in just one rapid step. This detailed meth- odology can be used for any protein. It gives us proteins with a good yield and in a short time.
In the present study and for the first time, glyceraldehyde-3-dehydrogenase was purified from human erythrocytes by immunoaffinity chromatography.
The comparison between the traditional method and our new procedure was done. Also, we report the characteriz- ation of the GAPDH purified in terms of native and subunit molecular weights, western blot analyses, and physicochemical and kinetic parameters.
Materials and Methods
Materials
DL-Glyceraldehyde-3-phosphate (DL-G3P) was prepared from monobarium salts of diethyl acetal (Sigma, St. Louis, USA). All other chemicals (analytical grade) were from Fluka (St. Gallen, Switzerland) or Merck (Darmstadt, Germany).
GAPDH purification
The enzyme was purified to electrophoretic homogeneity from human erythrocytes by two procedures; the first (traditional procedure) was based on ammonium sulfate fractionation and Blue Sepharose CL-6B chromatography [16 – 18], and the second (immunological procedure) was based on immunoaffinity chromatography. All steps were performed at 48C. Centrifugations were carried out at 3000 g for 45 min.
Traditional technique
The human blood was washed several times with the physiological water (NaCl, 0.9%, w/v) to eliminate the plasma. Then, to the washed red blood cells five volumes of distilled water were added and kept at 48C during 10 min. The extract obtained was subjected to
protein precipitation in the 66 – 88% saturation range of ammonium sulfate. The final pellet was dissolved in a minimal volume of 25 mM Tris – HCl ( pH 7.5), contain- ing 2 mM EDTA and 10 mM 2-mercaptoethanol (buffer A). The protein solution was dialyzed overnight against 5 L of the same buffer. The dialyzed enzyme preparation was applied to a Blue Sepharose CL-6B column equili- brated with two bed volumes of buffer A. The column was washed with three bed volumes of buffer A and two bed volumes of the same buffer adjusted to pH 8.5 (buffer B). The enzyme was eventually eluted with buffer B containing 10 mM NADþ at a flow rate of 6 ml/h. Active fractions were collected and preserved in 50% (v/v) glycerol at2208C until use.
Immunological technique
GAPDH-Sepharose gel preparation Two grams of acti- vated CN-Br Sepharose (Pharmacia, Uppsala, Sweden) were put in 1 mM HCl and then equilibrated with PBS ( pH 7.4). Ten milligrams of the purified GAPDH were mixed with the gel in the PBS and incubated for 24 h at room temperature. At the end of the reaction, the gel was extensively washed with the PBS. The unsaturated sites were blocked by 1 M ethanolamine ( pH 8) for 2 h. The excess of ethanolamine was eliminated by successive washes with the PBS.
Anti-GAPDH antibodies production A New Zealand white rabbit (1.5 kg) was injected with 1 mg of the GAPDH purified with the traditional procedure in aqueous solution (v/v) with incomplete Freund’s adjuvant. After 21 days, a second dose of 800mg of GAPDH was injected. After 1 week, a third dose of 500mg was then injected. One week later, 50 ml of rabbit blood was collected and the serum was separated by letting it coagulate overnight at 48C and centrifuging it.
Anti-GAPDH antibodies purification The obtained serum, containing monospecific anti-GAPDH polyclonal anti- bodies, was brought to 40% saturation with solid ammonium sulfate, stirred for 1 h and then centrifuged at 3000 g for 45 min. Afterward, the pellet was dissolved in a minimal volume of phosphate buffer saline (PBS, pH 7.4) containing 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 4.3 mM K2HPO4. The antibodies solution was dialyzed overnight against 5 L of the same buffer. The dialyzed antibody preparation was applied at a flow rate of 6 ml/h to a DEAE-cellulose (Serva, Heidelberg, Germany) column (3 cm diameter12 cm height) that has been previously equilibrated with PBS.
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The column was extensively washed at the same flow rate with equilibrating buffer solution. Fractions of 2 ml were collected and those containing the anti-GAPDH antibodies were pooled. The anti-GAPDH antibodies leave generally with dead volume of the column because they are isoelectric at pH 7.4 and they are not retained by the DEAE-cellulose. All the fractions containing anti-GAPDH antibodies are pooled.
Then, total polyclonal antibodies were added to the GAPDH-Sepharose gel and were incubated overnight in 48C under weak agitation. The mixture was transferred into a column (1 cm diameter10 cm height), and the column was washed with an excess of PBS.
Anti-GAPDH polyclonal antibodies were eluted by 100 mM glycine ( pH 2.5). Fractions of 1 ml were col- lected in tubes containing 100ml of 1 M Tris – HCl ( pH 9) with the aim of an immediate neutralization of the pH. After the elution of antibodies, the column was regenerated with PBS for a possible reuse. Anti-GAPDH antibodies harvested were dialyzed against 5 L of PBS overnight and preserved at 2208C until use.
GAPDH purification by immunoaffinity
Anti-GAPDH antibody-Sepharose gel was prepared as described above. Two grams of activated CN-Br Sepharose were mixed with 30 mg of the purified anti-GAPDH antibodies in PBS buffer ( pH 7.4) and incubated for 24 h at room temperature. After saturation with ethanolamine, gels were washed with PBS.
The crude extract of the human erythrocytes prepared as described above (traditional procedure) was subjected to the affinity chromatography using the same procedure described for the purification of the antibodies. Blood extract was added to the anti-GAPDH antibody- Sepharose gel and was incubated overnight at 48C under weak agitation. The mixture was transferred into a column (1 cm diameter10 cm height), then washed with an excess of PBS. GAPDH was eluted by 5 M MgCl2( pH 7). Active fractions were collected, dialyzed overnight against 2 L of the buffer A, and preserved in 50% (v/v) glycerol at 2208C until use. Protein content was measured according to the Bradford procedure, using bovine serum albumin (BSA) as standard [19].
GAPDH activity determination
GAPDH activity in the oxidative phosphorylation was determined spectrophotometrically at 258C by monitor- ing NADH generation at 340 nm [20]. The reaction mixture of 1 ml contained 50 mM Tricine – NaOH buffer
( pH 8.5), 10 mM sodium arsenate, 1 mM NADþ, and 2 mM D-G3P.
GAPDH kinetic studies
Initial velocities of the enzymatic reaction were calcu- lated by varying the concentration of the substrates, G3P (from 0.1 to 2 mM) or NADþ (from 0.1 to 2 mM).
Michaelis (Km) and dissociation (KD) constants, and the maximal velocities for the oxidation of G3P and the reduction of NADþ by the GAPDH were calculated by mathematical analysis according to the method of Cleland [21].
Determination of optimal pH and temperature-dependent GAPDH activity
The effect of pH on GAPDH activity was studied in a pH range from 4 to 10 using a mixture of different buffers (Tris, Mes, Hepes, potassium phosphate, and sodium acetate). On the other hand, thermal activation experiments were carried out by incubating the assay mixture for 5 min at temperatures ranging from 20 to 658C. Thermal denaturation was determined by measur- ing activity of the purified enzyme incubated for 5 min at temperatures ranging from 20 to 658C.
Denaturing polyacrylamide gel electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophor- esis (SDS-PAGE) was performed as described by Laemmli [22] on 12% one-dimensional polyacrylamide slab gels containing 0.1% SDS. Gels were run on a min- iature vertical slab gel unit (Hoefer Scientific Instruments, Holliston, USA). After electrophoresis, gels were stained with 0.025% (w/v) Coomassie Brilliant Blue R-250 in methanol/acetic acid/water (4:1:5, v/v/v) for 30 min at room temperature. Distaining was done in methanol/acetic acid/water (4:1:5, v/v/v). The apparent subunit molecular weight was determined by measuring relative mobility and by comparing with the pre-stained SDS-PAGE molecular weight standards (Sigma).
Native molecular weight determination
To determine the native molecular weight of the purified GAPDH, non-denaturing polyacrylamide gel electrophor- esis was carried out according to the method of Hedrick and Smith [23]. The separating gels (6, 8, 10, and 12%
polyacrylamide) were buffered with 1.5 M Tris–HCl ( pH 8.8). The running buffer was composed of 25 mM Tris and 320 mM glycine ( pH 8.6). All experiments were realized at 48C. The electrophoresis running conditions, staining and distaining, were as described for SDS-PAGE.
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The relative molecular weight of the native purified GAPDH was estimated using native molecular weight markers (Sigma): dimeric and monomeric urease (545 and 272 kDa), dimeric and monomeric BSA (132 and 66 kDa), and ovalbumin (45 kDa). By constructing the Ferguson plot [(log (Rf100) vs. the concentration of polyacryl- amide gels (%)], the resulting slopes vs. the standard native proteins of known molecular weight, permit the determi- nation of the molecular weight of purified GAPDH.
Western blotting
After SDS-PAGE (12%) and subsequent transfer in nitrocellulose [24], the proteins (50mg) were exposed to 1:100 dilution of monospecific polyclonal anti-GAPDH antibody and detected with the secondary antibody of anti-rabbit, IgG-Horseradish peroxidase conjugate (1:2500) (Promega, Madison, USA). The visualization is made in darkness using the following mixture: 12 mg of 4-chloro-1-naphtol in 4 ml of methanol, 16 ml of PBS, and 100ml of 30 % H2O2.
Statistical data analysis
In each assay, the experimental data represent the mean+SD (three independent assays). Means were compared using the Student’s t-test. Differences were considered significant at the levelP,0.05 and very sig- nificant at the levelP,0.01.
Results
Purification of human GAPDH
The GAPDH was purified to electrophoretic homogen- eity from human erythrocytes by two procedures:
In the first procedure, a total amount of about 1133 mg of protein, corresponding to approximately 17 units of GAPDH, was obtained from a crude extract of human erythrocytes. The purification of the enzyme was performed by ammonium sulfate precipitation, followed by Blue Sepharose CL-6B chromatography, which was carried out at 48C as described previously [16 – 18]. After
the precipitation by ammonium sulfate which facilitated the elimination of more than 76.7% of contaminating proteins, the concentrated enzyme solution was applied to a Cibacron blue-agarose column.Table 1summarizes the representative purification protocol. Indeed, values of 0.79 U/mg of protein were obtained for the purified enzyme with a yield of 17.7% and a purification factor of approximately 52.8 fold.
The SDS-PAGE analysis of different fractions obtained during this purification procedure showed a progressive enrichment in 36 kDa protein [Fig. 1(A)]. Only this protein band, having the same size as the GAPDH subunit, was seen in the electrophoretically homogeneous final enzyme preparations [Fig. 1(A), lane 3)].
We have produced rabbit polyclonal antibodies using purified human GAPDH as immunogen. These anti- bodies selectively reacted by the immunoblotting pro- cedure with a single immunoreactive band in both crude extracts and purified preparations. Fig. 1(B) shows that the relative molecular mass of the detected protein (36 kDa) is the expected one for the GAPDH monomer.
The anti-GAPDH antibodies produced were purified to electrophoretic homogeneity from rabbit serum (Fig. 2). Then, the purified polyclonal antibodies were immobilized onto CNBr Sepharose, which is used to purify the GAPDH in one step (second procedure).
Indeed, for this method, a total amount of about 957 mg of protein, corresponding to approximately 15.5 units of GAPDH, was obtained from the crude extract of human erythrocytes. Values of 1.1 U/mg of protein were obtained for the purified enzyme with a yield of 68.7% and a purification factor of approximately 68.7 fold (Table 2).
The analysis of the different fractions obtained during this purification procedure showed also a significant enrichment in 36 kDa protein [Fig. 3(A)].
The results obtained showed that the two procedures used facilitated the purification of GAPDH, however, the second method presents a high purification factor and gives an significant quantity of purified GAPDH.
Table 1 Purification of the GAPDH from human erythrocytes by conventional method
Total protein (mg) Specific activity (U/mg protein)
Total activity (U) Purification factor (fold)
Yield (%)
Crude extract 1133 0.015 17 1 100
Ammonium sulfate (66 – 88%) 90 0.044 3.96 2.9 23.3
Sepharose-Blue 3.8 0.793 3.01 52.8 17.7
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Physicochemical and kinetic properties of human GAPDH
The molecular weight of the purified GAPDH subunit was estimated by plotting using molecular weight marker proteins [Fig. 4(A)]. The weight obtained corre- sponds to 36 kDa.
To determine the molecular weight of the native enzyme, an electrophoresis in a non-denaturing system was performed using different separating gels (6, 8, 10, and 12% polyacrylamide). From the Ferguson plot [Fig. 4(B)], a value of 150 kDa was estimated for the molecular weight of the native GAPDH. This result, compared with that obtained from SDS-PAGE, which shows a single band corresponding to 36 kDa protein [Fig. 1(A), lane 3], suggests that GAPDH purified from human erythrocytes should have a homotetrameric struc- ture like other GAPDHs [2].
Studies on the effect of temperature on GAPDH activity revealed an optimal value of 40 – 458C [Fig. 5(A)]. The optimal pH value for the oxidative reac- tion was 8.5 [Fig. 5(B)].
Initial velocities of the enzymatic reaction were calcu- lated by varying the concentration of the substrates, G3P (from 0.1 to 2 mM) or NADþ (from 0.1 to 2 mM).
Values of the Michaelis constants (Km) and dissociation constants (KD), and the maximal velocities for the oxi- dation of G3P and the reduction of NADþ by the GAPDH were obtained by mathematical analysis. The KmNADþ, KmG3P, and KDNADþ were estimated to be 17.8+0.8, 20.7+0.7, and 205+6mM, respectively.
The Vmax of the purified protein was estimated to be 4.3+0.14 U/mg.
Discussion
The GAPDH was purified to electrophoretic homogeneity from human erythrocytes by two procedures; the first pro- cedure was performed by ammonium sulfate precipitation, followed by Blue Sepharose CL-6B chromatography and the second procedure was done using CNBr Sepharose activated by anti-GAPDH polyclonal antibodies.
Fig. 1 GAPDH purification steps from human erythrocytes with traditional procedure Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (A) or subjected to western blotting (B) using the purified polyclonal antibodies anti-human GAPDH. Lanes M, 1, 2, and 3 represent standard proteins, crude extract, 66 – 88% ammonium sulfate fraction, and affinity chromatography eluate pool ( pure protein preparation). Bound antibody was located by coupled immunoreaction with peroxidase conjugated goat anti-rabbit IgG. The arrow (B) indicates the band corresponding to the GAPDH subunit.
Fig. 2 Purification steps of the anti-GAPDH antibodies Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. Lanes 1, 2, 3, and 4 represent crude serum, 25% (w/v) ammonium sulfate fraction, anion-exchange Chromatography eluate pool, and affinity chromatography eluate pool ( pure polyclonal antibody preparation).
Table 2 Purification of the GAPDH from human erythrocytes by immunoaffinity chromatography
Total protein (mg) Specific activity (U/mg protein)
Total activity (U) Purification factor (fold)
Yield (%)
Crude extract 957 0.016 15.5 1 100
Immunoaffinity column 9.6 1.10 10.6 68.7 68.3
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The SDS-PAGE analysis of different fractions obtained during the two procedures showed a progressive enrichment in 36 kDa protein and the western blotting showed that the purified protein corresponds to the GAPDH monomer (Figs. 1and2).
In the first procedure, from 1133 mg of total proteins, 3.8 mg of the purified GAPDH was obtained with a yield of 17.7% and a purification factor of approxi- mately 52.8 fold (Table 1). In the second procedure, from 957 mg of total proteins, 9.6 mg of the purified GAPDH was obtained with a yield of 68.7% and a purification factor of approximately 68.7 fold (Table 2).
As we can see, the two procedures used facilitated the purification of GAPDH, however, the second method presents a high purification factor and gives an signifi- cant quantity of purified GAPDH.
The molecular weight obtained for the purified protein corresponds to 36 kDa. Similar results were obtained for the GAPDH of Jaculus orientalis [16] andTetrahymena pyriformis [17]. ForBacillus cereus, the weight obtained was 35 kDa [18] and it was 37 kDa, for Pleurodeles waltl [25],Camelus dromedarius [26], and Sardina pil- chardus [27]. The molecular weight of the native enzyme was estimated at 150 kDa, suggesting that this
enzyme had a homotetrameric structure like other GAPDHs [2].
Studies on the effect of temperature on GAPDH activity revealed an optimal value of 40 – 458C [Fig. 5(A)]. Some differences were observed for other species. Indeed, it is equal to 308C forSardina pilchardus [27] and Camelus dromedarius [26], 358C for Jaculus orientalis[16] andTetrahymena pyriformis[17], 388C for Bacillus cereus[18], and 408C forPleurodeles waltl[25].
The optimal pH value for the oxidative reaction was 8.5 [Fig. 5(B)]. An identical value was obtained for the
Fig. 3 GAPDH purification steps from human erythrocytes with the immunological technique Proteins were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (A) or subjected to western blotting (B) using the purified anti-human GAPDH polyclonal antibodies. Lanes 1 and 2 represent crude extract and affinity chromatography eluate pool ( pure protein preparation).
Bound antibody was located by coupled immunoreaction with peroxidase conjugated goat anti-rabbit IgG. The arrow (B) indicates the band corresponding to the GAPDH subunit.
Fig. 4 Determination of molecular weight of the purified GAPDH (A) Molecular weight of the purified GAPDH subunit was estimated by plotting. Molecular weight marker proteins were BSA (66 kDa), ovalbumin (45 kDa), GAPDH (36 kDa), trypsin (24 kDa), trypsin inhibitor (20.4 kDa) and lysozyme (14.2 kDa). (B) Determination of the native molecular weight of the purified GAPDH by native gel electrophoresis of various concentrations of polyacrylamide (6, 8, 10, and 12%). Molecular weight marker proteins were dimeric and monomeric urease (545 and 272 kDa), dimeric and monomeric BSA (132 and 66 kDa), and ovalbumin (45 kDa). Relative mobilities of proteins plotted as Log (Relative mobility100) vs. gel concentration are indicated on inset. A plot of the obtained slopes vs.
molecular weight was linear and used to determine native GAPDH molecular weight.
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GAPDH ofJaculus orientalis[16],Tetrahymena pyrifor- mis[17],Bacillus cereus[18], andPleurodeles waltl[25].
However, for Sardina pilchardus, the optimal pH was 8 [27] and forCamelus dromedariusit was 7.8 [26].
Values of the Michaelis constants (Km) and dis- sociation constants (KD), and the maximal velocities for the oxidation of G3P and the reduction of NADþ by the GAPDH were calculated. The KmNADþ, KmG3P, and KDNADþwere estimated to be 17.8, 20.7, and 205mM, respectively. The Vmax of the purified protein was esti- mated to be 4.3 U/mg. For the purified GAPDH from Tetrahymena pyriformis, the Vmaxwas similar (5.6 U/mg) but the KmNAD and KmG3P were different (5 and 150mM respectively) [17], and for Pleurodeles waltl, they were 60 mM, 27mM, and 33.2 U/mg [25].
In conclusion, it is for the first time that the glyceralde- hyde 3-phosphate dehydrogenase has been purified from human erythrocytes with the highest yield in a short time.
The characterization of the native GAPDH reveals that it is a homotetramer of 150 kDa with absolute specificity
for NADþ. Analysis by western blots using purified monospecific polyclonal antibodies raised against the pur- ified GAPDH showed a single 36 kDa band correspond- ing to the enzyme subunit. Accordingly, studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 40 –458C and 8.5, respectively.
The kinetic parameters were also calculated: theKm value against G3P was 20.7mM and against NADþ was 17.8mM and the Vmax was 4.3 U/mg. Our protocol rep- resents a simple, economic, and reproducible tool for the purification of high quantity of human GAPDH. This method can also be used for any other protein.
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