Separation of Urinary Proteins and Diagnosis of Proteinurias

Diagnosis of pathological urinary profiles and estimation of the molecular size of the corresponding proteins is possible by SDS PA gradient gel electrophoresis under nonreducing conditions. Protein patterns may be estimated in micro-sized (43x50x0.45 mm) SDS gradient gels of 8-25% T fixed to a plastic backing (GelBond™) as they are

2.0 2.5 3.0 3.5 4.0 4.5 5.0

Figure 15 Migration distances of denatured proteins and protein subunits obtained by SDS PA gradient gel electrophoresis. The logarithm of the molecular mass of proteins (log Mr) is linearly correlated to the square root of the PA concentration T) which they reached upon electrophoresis. Also, log Mr is linearly correlated to the square root of the migration distance (^ D (D (mm)) which proteins reached upon electrophoresis. Reproduced with permission from Rothe (1994).

commercially available together with a suitable horizontal electrophoretic apparatus (Phast system) and an automated silver staining device (Amersham Pharmacia Biotech). The method has the advantage that urine samples need not be concentrated or desalted before electrophoresis. Samples may be stored frozen at — 20°C after addition of sodium azide and after particulate removal by centrifugation. Samples with protein concentrations above 0.30 mg mL"1 must be diluted. Proteins must not be reduced (e.g. with 2-mercaptoethanol) since under SDS and nonreducing conditions the quarternary structure of all major serum proteins excreted in urine is unaffected, except haemoglobin which is split into its monomers and dimers. Figure 16 shows some selected protein patterns of renal malfunctions.

Table 13 Gel and buffer system used to separate small peptides in SDS PA gradient gel electrophoresis

Acrylamide (g 100 mL~1)

Gel buffer (pH)

Electrode buffer (pH) Correlation Authors (Mr (Da) range)

10-18 0.5-0.9 Stacking gel: 5% acrylamide,

0.13% Bis, 0.067 mol L~1 Tris-HCl, pH 6.8, 0.1% SDS, 0.067% ammonium persulfate and 0.067% TEMED; separation gel: 0.45 mol L~1 Tris-HCl, pH 6.9, 0.1% SDS, 0.05% ammonium persulfate, 0.05% TEMED, 7 mol L~1 urea

0.05 mol L~1 Tris, 0.38 mol L^1 glycine, 0.1 % SDS, pH 8.5

Hashimoto etal. (1983), Laemmli (1970)

Mr (Da), mol mass; D(mm), migration distance; TEMED, A/,A/,W,W-tetramethylethylenediamine. The buffer solution containing 10% acrylamide (0.5% Bis) contains no sucrose while the buffer solution containing 18% acrylamide (0.9% Bis) contains 10% (w/v) of sucrose. The PAA concentration and the sucrose concentration increase linearly from top to bottom. The system can also be used to separate lipopolysaccharidesand phospholipids. The addition of iodoacetamideto samples prior to electrophoresis eliminated artifacts currently observed in silver staining of protein bands. Log Mr correlates linearly with migration distance (D(mm)) in the mon mass range of 1.4 (kDa) to 17 (kDa). Flat gels of the dimensions 150 x 140 (height) x 1 (mm) were used. Gels were run for at least 15 h at 120 V. Samples were heated for 2 min at 100°C in a sample buffer containing 10% sucrose, 0.0625 M Tris-HCl, pH 6.8, 2% SDS, 10 mM dithiothreitol and 0.0025% Bromophenol blue (if necessary they were treated with iodoacetamide). Reproduced with permission from Rothe and Maurer (1986).

Table 14 Mol masses of polypeptides and peptides employed for urea-SDS gel electrophoresis

Protein

Mol mass (Da)

Literature valuea

Computedb

Ovalbumin

46000

Carboxypeptidase A

34500

c

Myoglobin

17200

Myoglobin I # II

14900

Cytochrome c

12300

c

Myoglobin I

8270

Cytochrome c I

7760

Myoglobin II

6420

Bovine trypsin inhibitor

6160

c

Adrenocorticotrophic hormone

4550

6500

Insulin

5700

Insulin B chain

3400

Insulin A chain

2300

Glucagon

3460

1800

Cytochrome c II

2780

Myoglobin III

2550

Cytochrome c III

1810

Bacitracin

1400

Polymyxin B

1225

2200

aValues as cited by Swank and Munkres (1971). bValues calculated by Swank and Munkres (1971) using least-squares regression analysis and assuming a linear correlation between log Mr (Mr, mol mass (Da)) and migration distance D(mm).

cThe mol masses of these proteins also deviate considerably if a straight line in a log Mr vs. Dplot is drawn through the points of carboxypeptidase A and bacitracin.

References as given in Rothe and Maurer (1986). Reproduced with permission from Rothe and Maurer (1986).

Diagnosis of the following proteinurias is possible:

1. Proteinuria in the normal range of total protein

2. Orthostatic (postural) proteinuria

3. Post-renal proteinurias

(a) Post-renal haematuria

(b) Local excretion of proteins

4. Bence-Jones proteinuria

5. Lower and upper urinary tract infections: cystitis and pyelonephritis

6. Diabetes mellitus

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