Glycoproteins and Mucoproteins

Conjugated proteins containing a complex carbohydrate moiety linked to the protein chain are referred to as glyco- and mucoproteins. A large number are known with considerable individual variation in composition and properties. The carbohydrate chain linked to the protein is made up of the hexoses, galactose; the methylpentose, fucose; and a sialic acid, such as N-acetylneuraminic acid. The sialic acid is located at one end of the carbohydrate chain; the other end of the chain is linked by a peptide bond to one of the peptide chains of the protein molecule.

In a useful classification, those carbohydrate-proteins containing less than 4 per cent of hexosamines are termed glycoproteins. They contain from a trace up to about 15 per cent of total carbohydrate. Those with over 4 per cent of hexosamines and containing from 10 to 75 per cent of carbohydrate are termed mucoproteins. The expression mucoids is also used in characterizing those mucoproteins in which the bond linking the carbohydrate chain and the protein is split only with difficulty, in contrast to another group containing acidic mucopolysaccharides, in which the linkage is polar and easily split. The glyco- and mucoproteins differ from the albumins and globulins in that they are not precipitated by perchloric and sulfosalicylic acids.

Mucoproteins of clinical interest are found in both serum and urine. Electrophoretically, most mucoproteins migrate with a mobility associated with alpha-1 globulins, although individual proteins may be found among all the usual electrophoretic groups. They are identified on electrophoretic strips or gels by their capacity to give a positive staining reaction with Schiff's reagent. The characteristic pink color is due to a reaction between the carbohydrate portion of the mucoprotein and the reagent. The mucoid present in serum in greatest amount is orosomucoid; it has a molecular weight of about 40,000 and is quite acidic, possessing an isoelectric point at pH 2.3. A number of other mucoproteins are also present in varying amounts.

Amino Acids and Related Metabolites

Next to urea, amino acids constitute the second largest source of the nonprotein nitrogen (NPN) in serum. In general, serum urea-N and serum NPN parallel each other in serum; as the former rises, the latter increases in about the same proportion.

Amyloid

As a consequence of long term, chronic, suppurative disease and a number of other conditions there is deposited in the blood vessels and matrix of a number of organs a type of abnormal proteinaceous material called amyloid.

Albumin determination by dye binding

All proteins, and especially albumin, tend to react with many chemical species by means of electrostatic and tertiary van der Waal's forces and by virtue of hydrogen bonding. Bilirubin, fatty acids, most hormones, and many drugs are transported about the body bound to albumin. Many colored dyes in the anionic form also possess this protein-binding property. This property has been used in attempts to devise methods by which albumin could be measured directly, without previous precipitation of globulins.

In dye-binding methods, only those dyes or indicators can be used that bind very tightly to the albumin molecule, so that practically 100 per cent of the albumin present is bound to dye. The binding must be unaffected by reasonably small changes in ionic strength and pH. Further, the color of the protein-bound dye should be different from that of the free dye; i.e., there should be a substantial shift in the wavelength of light at which maximum absorption occurs in the two forms. The albumin-dye concentration can then be measured in the presence of excess dye. Finally, dye binding to other protein fractions (globulins) must be negligible if the dye binding is to be the basis for a valid assay of albumin. For practical reasons, the color characteristics of the dye should be such that it can be measured at wavelengths of light where bilirubin and hemoglobin will give negligible or minimal interference. The use of methyl orange was proposed by Bracken and Klotz and Watson and Nankiville; HABA (2-(4'-hydroxyazobenzene)-benzoic acid) by Rutstein, Ingenito, and Reynolds and Martinek, and bromcresol green by Rodkey. The use of bromcresol green is most free of pigment interference. Bilirubin at levels over 5 mg./100 ml. interferes with HABA procedures and to a lesser degree with methyl orange methods.

The use of these dye reagents is now well established, and several manual and AutoAnalyzer procedures using methyl orange and HABA are available; however, there is considerable disagreement over the reliability of the dye-binding methods. Occasionally, albumin values obtained are unusually high as a result of the binding of the dye to proteins other than albumin. As an example of dye-binding technique, a methyl orange procedure will be presented in outline. It is a modification of the method proposed by Wrenn and Feichtmeir and is designed for use with the Coleman spectrophotometer and 19 mm. cuvets.

Assay of Serum Albumin by Methyl Orange Binding
Reagents

1.  Working dye reagent. Prepare by adding 5 to 10 ml. portions of 0.10 per cent (w/v) methyl orange to 1000 ml. of citrate buffer, 0.055 M. pH=3.50 +/- 0.03; check the absorbance against water at 540 nm. after each addition. Continue addition of dye until the absorbance lies between 0.78 and 0.85. Use the reagent at 24 to 27 degree of Celcius, although it is stored in the refrigerator.
2. Standards. These are prepared from crystalline human serum albumin. Bovine albumin should not be used because there are significant differences in dye binding by albumins from different species. It is wise to assay the albumin standard against the biuret total protein method so that the total protein and albumin values will be consistent. Prepare the stock reagent, which has a concentration of 10.00 gm./100 ml., by dissolving the albumin in saline. Prepare the working standards by diluting the stock with saline to give levels of 1.0 through 6.0 gm./100 ml.

Procedure

1.  Pipet 10.0 ml. of working dye reagent into a series of cuvets. One cuvet will be needed for the albumin blank, one each for the six standards, and one for each of the unknowns. Transfer 0.20 ml. each, of saline, standards, and sera, to the respective cuvets. After mixing by gentle inversion, permit the cuvets to stand for 20 minutes.
2. Read the absorbances of the standard and serum specimens against the blank at 540 nm. If the specimens are slightly lipemic, icteric, or hemolyzed, pigment color corrections will have to be made. For this purpose, add 0.20 ml. of the test serum to 10.0 ml. of the citrate buffer and read the absorbance against water (or citrate buffer) at 540 nm. Subtract these readings from the previous readings.
3. Convert the absorbance readings to albumin concentration by use of the calibration curve, which follows Beer's law up to concentrations of about 4 to 5 gm./100 ml. Prepare new calibration curves daily.

 Comments
Normal globulins and most abnormal globulins have no effect on the albumin values. Occasionally paraproteins and macroglobulins will interfere by giving high results. Dye binding, being a dissociation-association equilibrium phenomenon, is influenced by changes in temperature; a rise in temperature increases the dye-albumin dissociation and thus gives lower results. Therefore, standards and sera should be run under identical conditions. Heparinized plasma should not be used because heparin, for reasons not understood, enhances dye binding by albumin. Serum albumin levels obtained by methyl orange dye binding techniques tend to be 0.1 to 0.2 gm./100 ml. higher than those obtained by salt-fractionation or electrophoresis, although some chemists have reported results as much as 0.5 gm./100 ml. higher.

Qualitative tests for urine protein

In devising qualitative tests for urine proteins, it is desirable that the test be negative whenever protein is present in normal concentrations, but that the test be positive whenever protein concentration is greater than 20 to 25 mg./100 ml. The test should not be sensitive to mucins and other proteins of nonrenal origin and it should be capable of rough quantitation. A large number of such tests are available. The older tests are based either on precipitating the proteins by heat or by reaction with anionic protein precipitants. A more recent test depends on a change in the color of an indicator in the presence of protein.

Qualitative tests are best done on fresh morning specimens of urine. Such specimens are usually fairly concentrated, thus making possible the detection of proteinuria early in disease. They will also be free of the orthostatic effect discussed previously. If urines must be held for examination at a more convenient time, and if timed specimens are to be examined, they should be refrigerated and examined within 48 hours. Layering with toluene may be used to minimize microbial growth. Bacterially contaminated specimens (pyelonephritis, lower genitourinary tract infections) should be examined only when fresh.

Heat Coagulation Test

The pH of the urine is checked with bromcresol green pH indicator paper, (pH range, 3.8 to 5.5) and if above pH 5.0, acetic acid, 30 per cent (w/v) is added dropwise until the pH is between 4.0 and 4.6. If any turbidity or insoluble matter is present or is formed (mucoproteins may precipitate on adding the acid), it is removed by centrifugation. About 7 to 10 ml. of clear urine are placed in a 15 x 120 mm. test tube and the upper one-third is gently heated over a Bunsen flame until boiling just begins. The heated part is compared in ordinary light with the lower unheated portion of the specimen. Any turbidity, cloudiness, or precipitate demonstrates the presence of protein. The degree of the reaction is customarily graded 0 to ++++. False positive results may occur and are discussed in the section on interpretation of results.

Sulfosalicylic Acid Test
Into a 15 x 120 mm. test tube, place 5 ml. of clarified urine (pH 4.5 to 6.5). Then 0.50 to 0.80 ml. of 20 per cent (w/v) sulfosalicylic acid is added carefully down the sides of the tube to layer underneath the urine. After 1 minute the interface is examined for the presence or absence of turbidity or cloudiness. A barely evident turbidity (5 to 10 mg./100 ml) is graded as +/-, and increasing degrees of turbidity, cloudiness, or precipitation are graded + to ++++. False positives, due to nonspecific precipitation, may be encountered and are discussed in the section on interpretation of results.

The reagent is prepared by dissolving 100 gm. of reagent grade sulfosalicylic acid (2-hydroxybenzoic, 5-sulfonic acid dihydrate) in about 350 ml. of water with the aid of heat. The warm solution is filtered through two layers of filter paper and the total filtrate diluted to 500 ml. when cool. The solution should be crystal clear and colorless. It is discarded when turbidity or darkening develops.

Macroglobulins

The majority of plasma globulins have a molecular weight in the range of 150.000 to 200.000. A small proportion, less than 4 to 5 per cent of the total serum proteins, are very large size molecules, with molecular weights in the neighborhood of 1,000,000. These large proteins are referred to as macroglobulins. If plasma is centrifuged at ultrahigh speeds, three classes of proteins can be differentiated as a result of differences in the rate of sedimentation (see the section on ultracentrifuge separations).

The macroglobulins constitute the heaviest, S19. class, and they include 50 to 70 per cent of the alpha-2 globulins and 10 to 30 per cent of the gamma globulins. The normal level present in plasma is about 0.20 gm./100 ml., ranging from 0.07 to 0.43 gm./100 ml. In a very small number of individuals, considerably higher levels may be found. In primary macroglobulinemia, first studied in detail by Waldenstrom, the level of S19 proteins is well over 15 per cent of the total serum proteins. Macroglobulinemia is a metabolic disease, characterized by weight loss, and susceptibility to infection, and involves the entire reticuloendothelial system. The  serum electrophoretic pattern is similar to that found in multiple myeloma: Bence-Jones proteins are occasionally found, and some of the macroglobulins behave like cryoglobulins, with resultant impaired blood flow in the extremities. Secondary macroglobulinemia is more common, and is associated with a large number of different disease entities. The level of macroglobulins is always under 15 per cent. Secondary macroglobulinemia is found in patients with leukemia, lymphosarcoma, and rheumatoid arthritis. In myelomatosis, the concentration of S19 proteins is usually normal, or only slightly elevated.

Macroglobulinemia is best diagnosed by ultracentrifugation of a serum specimen. The peak demonstrating the presence of the high molecular weight IgM globulins shows up quite clearly. Isolation of the macroglobulins can also be achieved by gel filtration on Sephadex-200, using either column or thin-layer chromatography. Immunoelectrophoretic patterns can provide supportive evidence, the increased level of macroglobulins being demonstrated by the presence of heavy precipitant arcs with anti-IgM antisera.

 The Sia test is a simple qualitative test for macroglobulins based on the fact that the IgM globulins have decreased solubility in water that contains a low concentration of salts. In one procedure, 0.10 ml. of serum is added slowly down the sides of a test tube into a solution of 5.0 ml. of 0.01 M phosphate buffer, pH 7.1. If macroglobulins are present at levels over 0.7 gm./100 ml. a flaky, birefringent precipitate is formed, which dissolves on the addition of a spatula tip of NaCl. Normal, myeloma, and arthritis sera give no precipitation, and lipemic sera only a hazy opalescence. Malaria and kala-azar sera produce a positive result.

Serum Protein Fractionation

Only two methods of separating serum protein components are sufficiently simple and convenient to be useful in routine work. Salt fractionation is easy to carry out, involves no special equipment, and, if done carefully, provides very useful clinical information. Commonly, only separation of albumin from the globulins as a group is carried out, but techniques are available for separating and quantitating the chief globulin components, especially gamma globulin. When more precise values of the protein fractions are desired, electrophoretic techniques are used. These require more skill and the use of special equipment, and they are more time consuming, although with cellulose acetate membranes, separations may be made in less than an hour. Ultracentrifugation, requiring an expensive instrument, is not suitable for routine use, but is especially useful in the study of lipoproteins and macroglobulins.

Salt Fractionation; Albumin-Globulin Ratio
Albumins are soluble in water, whereas globulins are not. If an electrolyte solution of low concentration (0.1 M) is added to a globulin, the latter will go into solution by virtue of the phenomenon of salting-in. The cations and anions of the electrolyte bind to reactive groups on the protein molecules and break the bonds holding the protein micelles together, allowing the individual protein molecules to undergo solution. The salt concentration of plasma, approximately 0.15 M, is ideally suited to maintain globulins in solution. As the salt concentration is increased to high levels (of the order of 2 M), the various globulins will again become insoluble and will precipitate by salting-out. The level of electrolyte needed to precipitate the various globulins will vary with the individual protein and electrolyte and will also depend on pH and temperature.

Ammonium sulfate was the first salt to be used and it is still used in protein and enzyme investigations. Those proteins precipitating in one-third saturated ammonium sulfate solution were termed euglobulins (true globulins). As the salt concentration is increased, the pseudoglobulins begin to precipitate out, and at concentrations near 50 per cent saturation, all globulins become insoluble, the proteins still in solution constituting the albumins. The use of ammonium sulfate for the isolation and assay of gamma globulins will be described later. The salt is not used in routine clinical work because it interferes with the biuret reaction and obviously cannot be used with the Kjeldahl nitrogen technique.

The only salts whose use has become established in routine clinical chemistry are sodium sulfate and sodium sulfite. The former (Na2SO4) was used as early as 1901, but its large scale use followed the work of Howe in 1921, who recommended the use of 22 per cent (w/v) sodium sulfate to precipitate globulins. Total protein was assayed, and, after removing globulins by filtration, the albumin fraction was measured in the filtrate, globulin being calculated by difference. In 1940 Kingsley introduced the use of ether as a means of separating the precipitated globulins from the soluble albumins, without resorting to the slow, tedious filtration. By carefully dispersing diethyl ether into the mixture of serum plus salt solution, the insoluble globulin micelles would entrap sufficient ether so that on centrifugation, instead of sedimenting as they would normally, the globulins would float above the albumin solution as a compact pellicle. With this type of procedure, serum albumin was found to range from 4.0 to 5.5 gm./100 ml., the globulin from 2.0 to 2.9 gm./100 ml., and the albumin/globulin ratio from 1.5 to 2.5 (average, 2.0).

With the development of Tiselius' moving boundary electrophoresis technique, it was logical to compare the electrophoretic albumin fraction with that separated by the use of Howe's 22 per cent sodium sulfate. It was immediately evident that the Howe technique yielded higher values because it included with the albumins, the alpha-1 and some of the alpha-2 globulins. Studies by Majoor and Milne showed that to free the albumin of all globulin fractions, the concentration of sodium sulfate must be of the order of 26 to 27 per cent (w/v) (1.9 M). Unfortunately, this concentration is beyond the solubility of sodium sulfate at room temperature (25 degree of Celcius), and the salt solution must be kept at 35 to 37 degree of Celcius to avoid crystallization of the salt on storage and during use. With the use of 26 per cent sodium sulfate, the albumin/globulin ratio is of the order of 1.1 to 1.8, averaging about 1.4; this agrees quite well with that obtained by electrophoretic methods. Other salts have also been employed. Campbell and Hanna used sodium sulfite, obtaining complete separation of globulins from albumin at 27 per cent (w/v) (2.2 M). This salt has the advantage of being more soluble than sodium sulfate, and, because it acts as a buffer, better pH control of the precipitation reaction is possible. Reinhold proposed the use of a mixture of sodium sulfate plus sodium sulfite and this procedure will be presented in detail.

By using varying concentrations of sodium sulfate (or sodium sulfite), it is possible to separate all of the main globulin fractions from one another. For example, Kibrick and Blonstein used 15 per cent (w/v) sodium sulfate to precipitate the gamma globulins and 26 per cent salt concentration to precipitate all globulins. The difference between these values gave the sum of alpha and beta globulins. By further use of 19 per cent sodium sulfate, which precipitated the beta- plus gamma globulins, the amount of alpha globulins could also be determined. Such fractionations are tedious, however, and not too satisfactory, especially when abnormal sera are used, and they have never become popular. The electrophoretic separation is simpler and considerably more accurate.