Carl-Bertil Laurell

Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies

Abstract A method is deseribed for quantitative analysis of proteins with a charge differing from that of the bulk of the immunoglobulins. The method utilizes the difference between the rate of electrophoretic migration of proteins and of their antibody complexes in agarose gel. The method has an error of a few per cent; it is rapid, suitable for serial analysis, and requires 2 to 0.5 μg protein antigen. The amount of antiserum needed is slightly less than with the radial immunodiffusion techniques.

The polymorphism of “prealbumins” and α1-antitrypsin in human sera

Abstract Discontinuous, acid starch gel electrophoresis followed by electrophoresis into agarose containing anti- α 1 -antitrypsin gave precipitation patterns which can be explained by seven codominant alleles in the human for the serum globulin α 1 -antitrypsin. Since this protein is the major protease inhibitor of plasma we propose Pi as an abbreviation when describing this polymorphic protein system.

The Electrophoretic α1-Globulin Pattern of Serum in α1-Antitrypsin Deficiency

Th e main constituents of the a,-fraction obtained on paper electrophoresis in a barbital buff er are α1-lipoproteins, orosomucoid (acid glycoprotein), Schmid’s (1953) and Schultze et al.’s α1-3.5 S glycoprotein (Schultze, Giillner, Heide, Schonenberger & Schwick 1955), which Schultze, Heide & Haupt (1962) recently proved to be identical with a,-antitrypsin (10–12). Th e variation, in heaIth and disease, of the a,-lipoproteins, or at least of lipids in the electrophoretic albuminα1 region, has received fairly wide attention (for references see Lindgren & Nichols 1960) (9). Th e electrophoretic α1-lipoprotein fraction migrates faster and spreads more, widely than most of the other components. Its electrophoretic behavior also varies with the duration of storage of the serum and with variations of the intermediary lipid metabolism. It is the heaviest normal al-component (about 300 mg per 100 ml) calculated as protein, but it is electrophoretically heterogenous and does not give rise to any well demarcated electrophoretic protein peak, which is evident on comparison of the paper and agar gel electrophoretic patterns after staining for lipids and proteins. Th e orosomucoid, which normally occurs in a concentration of about 75 mg per 100 ml (Goodman, Ramsey, Simpson & Brennan 1957) produces no demonstrable peak on paper or in agar gel electrophoresis, because it is partly masked by the slower part of the albumin fraction, and it stains only faintly with bromphenol blue (Sundblad & WallinNilsson 1962) (4, 15). Th e α1x component normally occurs in a concentration of about 30 mg per 100 ml (Schultze, Heide & Haupt 1962), which is not high enough to produce a distinct peak on paper electrophoresis (11, 13). Burtin (1960) has stated that the strongly antigenic 3.5 S a,-glycoprotein (α1Z, α1-antitrypsin) is the dominating normal α1-component. We have accepted this concept for two reasons. Th e precipitation maximum of the line formed by antiα1-antitrypsin corresponds to the α1-protein peak obtained on paper and agar gel electrophoresis with diff erent buff ers. It may be deduced from data given by Jacobson (1955) on the α1antitrypsin activity that if the molecular weight of the α1-antitrypsin is about 60,000, the mean normal concentration will be 0.18 g per 100 ml. Th is amount of protein is in accord with the intensity of the relatively sharply demarcated alpha-1-band obtained on paper electrophoresis. We cannot accept Burtin’s statement, based on the appearance of immunoelectrophoretic patterns, that the main al-component varies little in pathological sera. On the contrary, the α1-antitrypsin varies considerably in disease (Jacobsson 1955), which is in accordance with the observed variations of the electrophoretic α1-band (3). In this article we present some patients with a new type of dysproteinemia characterized by very pronounced α1-antitrypsin defi ciency. Th e sera were revealed as abnormal on routine inspection of the paper electrophoretic strips at the laboratory since the α1 pattern had an atypical confi guration with no distinct α1-band. Th e numerical values of the electrophoretic α1-fractions fell within or just below the lower range of the normal variation. Originally published in Scandinavian Journal of Clinical and Laboratory Investigation 1963, 15 (2): 132–140 reprinted with permission.

α-1 Antitrypsin phenotypes determined by isoelectric focusing of the cysteine-antitrypsin mixed disulfide in serum

Abstract α-1 Antitrypsin phenotypes were determined from isoelectric focusing of whole serum in thin-layer acrylamide gels. The results confirmed that the technique permits reliable diagnosis. We validated the results by crossed immunoelectrophoresis, the inhibition of trypsin, and enrichment through interchange with the recently recognized antitrypsin thiol group. High resolution of the focusing technique facilitated recognition that cysteine reduction of whole serum improved the sharpness of the cathodal antitrypsin fractions. We have demonstrated the presence of prominent protein bands not due to antitrypsin in the M 4 and M 6 regions of a serum of Pi Z type. The thiol enrichment technique should prove useful to increase diagnostic reliability of isoelectrofocusing of α-1 antitrypsin.

A zinc-dependent peptidase in prostatic organelles present in seminal plasma

The electrophoretic seminal plasma protein pattern changes obviously within a few hours if the plasma is not frozen after the ejaculation. Mainly cathodal proteins disappear. Addition of serine protease inhibitors has no apparent affect, but o-phenanthroline markedly retards the conversions seen on electrophoresis. The subcellular organelles in seminal plasma are shown to contain a zinc ion dependent peptidase with optimal activity at pH 7.8. Estimations of enzyme activity may be performed using the elastase substrates n-tert butyloxycarbonyl-L-alanine-paranitrophenylester (Boc(Ala)) and succinyl(alanine)3-paranitroanilide (Suc(Ala)3pNA). The enzyme is inactivated by addition of o-phenanthroline and is completely reactivated by the addition of zinc ions.

A simple two-step procedure for the simultaneous isolation of corticosteroid binding globulin and sex hormone binding globulin from human serum by chromatography on cortisol-sepharose and phenyl-sepharose

Abstract Application of human serum to a cortisol-21-hemisuccinyl-AH-Sepharose column at pH 5.5 followed by cortisol gradient elution gave two partially separated proteins almost free from other contaminating components. The two proteins were corticosteroid binding globulin (CBG) and sex hormone binding globulin (SHBG) according to their physico-chemical properties and steroid binding characteristics. The affinity of SHBG, which showed no binding of free cortisol, to the cortisol-Sepharose gel is unexplained. In the second step, which was hydrophobic chromatography on phenyl-Sepharose, CBG and SHBG were separated from each other and further purified. The two proteins were obtained more than 95% pure according to sodium dodecyl sulphate polyacrylamide gel electrophoresis and they both had unique NH2-terminal sequences. Yields, which were measured by electroimmunoassay, were in the cortisol-Sepharose step 65% and 30% for CBG and SHBG, respectively and in the phenyl-Sepharose step 85% and 60%, respectively. This simple two-step procedure is well suited for large scale purification of these two steroid binding proteins from human serum.

Analysis of plasma α1-antitrypsin variants and their microheterogeneity

Abstract A new agarose gel electrophoretic method is described which permits immunochemical phenotyping of α 1 - antitrypsin in acetate buffers of pH 5.15 provided that an internal standard is used as mobility reference. The microheterogeneity of each α 1 - antitrypsin variant is apparent from their separation into a series of protein bands. The small differences in charge are not caused by the sialic acid content and they persist after isolation of the fractions by preparative electrophoresis. The crossed immunoelectrophoretic pattern of α 1 - antitrypsin and its subfractions at pH 5.15 may be used for demonstrating any change in the microheterogeneity of α 1 - antitrypsin during isolation and storage.

The serum α1-antitrypsin in families with hypo-α1-antitrypsinemia

Abstract The serum content of α1-antitrypsin was estimated with immunoprecipitation in tubes utilizing a specific antiserum. The results agreed with those obtained in association with the determination of the total antitryptic activity of serum in members of families with hypo-α1-antitrypsinemia. No overlapping occurred between normals, hetero- and homozygotes. The non-α1-antitrypsins of 1 ml serum inhibited in the average 0.15 mg trypsin.

Chloroquine Treatment in Rheumatoid Arthritis

15 patients with active RA were observed over a 3-month period after starting chloroquine treatment. Clinical condition, plasma levels of chloroquine and levels of 15 individual plasma proteins were checked monthly. Nine patients responded favourably to therapy, 6 failed to respond. The responders had lower initial CRP, orosomucoid and ceruloplasmin levels, whereas their IgA and IgM levels were slightly elevated. Significant reductions in the levels of CRP, haptoglobin, orosomucoid, fibrinogen and ceruloplasmin occurred in the responder group of patients. Alfa1-antitrypsin, antichymotrypsin C3 and C4 levels within the normal range were frequently encountered despite other clear-cut signs of activity. The chloroquine levels did not differ between responders and non-responders, the mean concentrations being 1.04 and 1.6 micromol/l respectively. This study has also demonstrated that in selected cases, despite active joint disease, all acute phase proteins may be normal. Finally, it was obvious that chloroquine, even when inducing remission, only brought about a partial normalization of the plasma protein pattern.

Complexes in plasma between light chain κ immunoglobulins and α1-antitrypsin respectively prealbumin

Abstract Complexes between κ-chains and α 1 -antitrypsin are regularly demonstrable in plasma from myeloma patients excreting more than 1 g κ-chains per litre of urine. Prealbumin produces similar complexes. These complexes are sensitive for mild reduction with thiol reagents. No corresponding complexes are produced with λ-chains. Conditions are described for the formation of the κ - α 1 -antitrypsin complexes in vitro . Substitution of the terminal SH-group of the κ-chains prevents the formation of complexes as does addition of thiol reagents. A thiol-disulphide exchange seems probable between the ultimate SH-group of κ-chains and a disulphide in α 1 -antitrypsin. The absence of λ-chain complexes may partly be explained by a lower reactivity of their penultimate cysteinyl than that of the terminal κ-chain thiol. Complexes between IgA and α 1 -at are regular constituents of plasma. They constitute about one per cent of the IgA. Both IgAK and IgAL produce complexes.