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Prostate-specific antigen (PSA) is one of the three most abundant prostatic-secreted proteins in human semen. It is a serine proteinase that, in its primary structure, manifests extensive similarities with that of the Arg-restricted glandular kallikrein-like proteinases. When isolated from semen by the addition of chromatography on aprotinin-Sepharose to a previously described procedure, PSA displayed chymotrypsin-like activity and cleaved semenogelin and the semenogelin-related proteins in a rapid and characteristic pattern, but had no trypsin-like activity. About one third of the purified protein was found to be enzymatically inactive, due to cleavage carboxy-terminal of Lys145. Active PSA formed SDS-stable complexes with alpha 1-antichymotrypsin, alpha 2-macroglobulin-analogue pregnancy zone protein. PSA formed inhibitory complexes with alpha 1-antichymotrypsin at a molar ratio of 1:1, a reaction in which PSA cleaved the inhibitor in a position identical to that reported from the reaction between chymotrypsin and alpha 1-antichymotrypsin. The formation of stable complexes between PSA and alpha 1-antichymotrypsin occurred at a much slower rate than that between chymotrypsin and alpha 1-antichymotrypsin, and at a similar or slightly slower rate than that between PSA and alpha 2-macroglobulin. When added to normal blood plasma in vitro, active PSA formed stable complexes both with alpha 2-macroglobulin and alpha 1-antichymotrypsin. This complex formation may be a crucial determinant of the turnover of active PSA in intercellular fluid or blood plasma in vivo.

Plasma samples exhibiting alloalbuminemia on electrophoresis at pH 8.6 were requested from clinical laboratories throughout Sweden. Nine variants, each representing a different single point mutation, were found in 100 apparently unrelated Swedes. The overall prevalence of alloalbuminemia was estimated at 1:1700. Mutations were identified by protein-structural analysis followed by allele-specific DNA hybridization to verify the most common types. Slightly retarded (+1) mobility was seen in 80 cases. Of these, 71 had the Arg(-2)----Cys proalbumin variant previously called Malmö I proalbumin. Thirteen examples of the second most frequent type, the substitution Lys313----Asn and a mobility change of -1 charge unit, were found, as well as six cases of Glu570----Lys (albumin B) and a single case of Arg-1----Gln (proalbumin Christchurch). Five previously unreported types of alloalbuminemia were identified: four instances of Glu376----Gln, which is the second known mutation at this site; two examples of Asp550----Ala, the second mutation reported at this site; and one example each of Asp63----Asn, Gln268----Arg, and Asn318----Lys. Other mutations were identified among eight subjects of foreign descent. The high frequency and relatively uniform geographic distribution of the Arg-2----Cys mutation suggest that it may have occurred in a founder individual many generation ago in Sweden.

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [Löbermann, H., Tokuoka, R., Deisenhofer, J.Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X.Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.

An electrophoretically slow albumin variant was detected with a phenotype frequency of about 1:1000 in Sweden and was also found in a family of Scottish descent from Kaikoura, New Zealand, and in five families in Tradate, Italy. Structural study established that the major variant component was arginyl-albumin, in which arginine at the -1 position of the propeptide is still attached to the processed albumin. A minor component with the amino-terminal sequence of proalbumin was also present as 3-6% of the total albumin. After amplification of the gene segment encoding the prepro sequence of albumin, specific hybridization of DNA to an oligonucleotide probe encoding cysteine at position -2 indicated the mutation of arginine at the -2 position to cysteine (-2 Arg----Cys). This produced the propeptide sequence Arg-Gly-Val-Phe-Cys-Arg. This was confirmed by sequence analysis after pyridylethylation of the cysteine. This mutation produces an alternate signal peptidase cleavage site in the variant proalbumin precursor of arginyl-albumin giving rise to two possible products, arginyl-albumin and the variant proalbumin. Another plasma from Bremen had an alloalbumin with a previously described substitution (1 Asp----Val), which also affects propeptide cleavage. Hypermutability of two CpG dinucleotides in the codons for the diarginyl sequence may account for the frequency of mutations in the propeptide. Mutation at these two sites results in a series of recurrent proalbumin variants that have arisen independently in diverse populations.

1. Monomeric nu-chains were conjugated with CNBr-activated Sepharose 4B. The C-terminal cysteine of the conjugated nu-chain was converted to a mixed disulfide with 3-carboxy-4-nitro-benzenethiol (Nbs) and used to separate plasma proteins with reactive thiol groups. The plasma proteins, alpha1-antitrypsin and prealbumin have the greatest affinity for the interchange reaction with mixed disulfides. The disulfide link between alpha1-antitrypsin and nu-chain is sensitive to excess Nbs, and is selectively cleaved in the presence of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) which accepts the sulfhydryl group of alpha1-antitrypsin. 2. A Simple method developed for the isolation of human alpha1-antitrypsin was equally effective for the various inherited phenotypes and for alpha1-antitrypsin from the dog, baboon, and monkey, Glutathione-Sepharose was also used successfully, but the nu-chain conjugate yielded alpha1-antitrypsin less contaminated with mercaptalbumin and prealbumin. 3. The alpha1-antitrypsin is harvested from this procedure as a mixed disulfide with Nbs. The negative charge of Nbs at pH 8.1 causes an increased electrophoretic mobility of the alpha1-antitrypsin derivative. Mild reduction liberates Nbs and electrophoretic mobility of alpha1-antitrypsin returns to normal. The method described can increase the alpha1-antitrypsin content of a plasma fraction from 5% of the total protein to 95% within one day with a yield of about 50%. This purification procedure does not exert any detectable effect on microheterogeneity.

The sequence of alpha 1-antitrypsin is in keeping with its role as a tissue scavenger of leukocyte elastase. Two abnormal variants commonly present in Europeans cause a deficiency that predisposes them to a progressive loss of lung elasticity. The nature of the reactive centre helps explain why cigarette smoking greatly accelerates the onset and severity of this degenerative process to give the disease emphysema.

The earlier observed pronounced hypogammaglobulinemia in germfree rats of different ages has been confirmed. Using an immunologic technique the concentration of immunologic gamma globulins were found to vary between 10 and 15 per cent of the values observed in ordinary rats. Upon contamination of germfree rats with the normal microbial flora a pronounced lag phase was noted before the gamma globulin level became normal. This lag phase was most pronounced in growing rats. Newborn rats seem to start gamma globulin production more rapidly than older germfree rats. The response with regard to gamma globulin production on contamination of germfree rats with different types of bacterial cells through the natural routes is not identical.

The main constituents of the a,-fraction obtained on paper electrophoresis in a barbital buffer are α1-lipoproteins, orosomucoid (acid glycoprotein), Schmid's (1953) and Schultze et al.'s α1-3.5 S ...

A short survey is given of the literature on haptoglobin, the hemoglobin-binding serum protein, its properties and biologic variations. The principles of an electrophoretic method for quantitative determination of the serum haptoglobin are described. Electrophoretic studies showed that haptoglobin has a high affinity for hemoglobin at physiologic pH and that every haptoglobin molecule can bind at least 2 hemoglobin molecules. Observations made following the intravenous injection of hemoglobin showed: that hemoglobin administered intravenously is bound by the haptoglobin; that free hemoglobin is not demonstrable until more hemoglobin has been injected than can be bound by the haptoglobin; that the complex hemoglobin-haptoglobin is eliminated from the plasma after intravascular hemolysis or intravenous administration of hemoglobin without being excreted in the urine; that the hemoglobin-haptoglobin complex is removed from the plasma at a constant rate during the major part of the elimination period; that the haptoglobin level will fall to nil within 24 hours, if the amount of hemoglobin injected is sufficient to bind all the haptoglobin available. During the following days the rate of formation of haptoglobin can be studied. From the data available it can be concluded that hemoglobinuria cannot appear until the amount of hemoglobin administered intravenously or the amount liberated intravascularly exceeds the binding power of the haptoglobin and the reabsorption capacity of the tubules. The variation observed by earlier authors in the so-called renal threshold for hemoglobin on intravenous injection of hemoglobin can be explained among other things by the variation in the haptoglobin content in one and the same subject, i.e., if the haptoglobin level is low, the threshold value will also be low, and vice versa.

Cumulative knowledge of the protein composition of plasma and newer techniques for specific analysis of proteins have made obsolete most flocculation tests, the albumin:globulin ratio, scanning diagrams, and most determinations of electrophoretic fractions. More clinically relevant information about the serum protein composition is obtained by critical visual evaluation of the protein bands obtained after electrophoretic separation of plasma or serum in nonadsorptive supporting media, if supplementary specific analysis is made of a small number of proteins such as albumin, orosomucoid, haptoglobins, ceruloplasmin, and the immunoglobulins of the three predominant classes

The electrophoretic fractions of serum proteins were studied up to the 5th generation in a colony of germ-free rats. The germ-free animals had significantly lower concentrations of beta and the gamma globulins, while the other fractions were within normal limits. Assuming that the gamma globulins survive equally long in the circulation in germ-free animals as in controls, the production of gamma globulins in normal rats is three times as rapid as in germ-free rats. This suggests that the normal flora of microorganisms is an important stimulant for the gamma globulin-producing cells.

A high correlation (r = 0.85) normally exists between the concentration of albumin and of IgG in cerebrospinal fluid (CSF). The correlation is still better (r = 0.96) if the concentrations of the two proteins are also measured in plasma and the CSF/ plasma ratios compared for albumin and IgG. The scatter diagram for these ratios in persons without disease of the nervous system is most useful as a reference when differentiating between local IgG production in the subarachnoid space and an increase in CSF protein for other reasons. The analysis can be performed on less than 0.1 ml of CSF, in contrast to agarose-gel electrophoresis, for which 5-10 ml is necessary.

1. A two-step-method is described by which human and canine prealbumin have been isolated from serum in good yield and high purity. The first step involves binding and release of prealbumin by a thiol-disulfide interchange reaction with thiol-Sepharose 4B. The second step consists of reversible binding of prealbumin to retinol-binding protein covalently linked to Sepharose 4B. 2. The canine prealbumin was immunochemically identified as an alpha2-globulin. Like human prealbumin it is a tetramer with some 10% smaller subunits than human prealbumin. Both proteins are devoid of carbohydrate and both bind thyroxin.

α1-Antitrypsin has been isolated from individuals with inherited genetic variants M3, X and Z. A fragmentation and peptide mapping system is described which together with amino acid and sequence analyses revealed the substitutions in M3 at 376 of Glu to Asp, in X at 204 of Glu to Lys and in the physiologically innocent Z a mutation at 213 of Val to Ala. The latter represents a second amino acid substitution in the Z protein.

CONTAMINATION of serum with neuraminidase-producing bacterial cells promptly produces changes in the electrophoretic pattern. The mobility (surface charge) of the α1-globulins (orosomucoid and α1-antitrypsin), of some α2-globulins (for example, haptoglobin) and of the β1-globulin, transferrin, decreases on release of sialic acid from the molecular surface1. Vulpe2 has published electrophoretic protein patterns of rabbit sera before and during development of experimental encephalomyelitis induced by injections of a brain suspension in combination with Freund's adjuvant. The changes in serum presented are similar to those produced by neuraminidase. Furthermore, the transferrin of human cerebrospinal fluid is electrophoretically heterogeneous3, indicating that part of it has lower sialic acid content than the most common transferrin type of Caucasians. These two observations prompted the present investigation of mammalian brain for neuraminidase. This enzyme is common among microorganisms, and has been found in chick-embryo chorioallantois but not in mammalian tissue4.

The comparison of measurements of fibronectin and lactoferrin in ejaculates from vasectomized men, subjects with functional deficiency or aplasia of the seminal vesicles, and reference subjects provided evidence that both the fibronectin and the lactoferrin in human seminal fluid originate from the seminal vesicles and the ampullae. The fibronectin is incorporated in the framework of the seminal gel formed during the immediate postejaculatory phase, whereas the lactoferrin remains in solution. In the seminal gel fibronectin is linked to its predominant structural protein, a high molecular weight seminal vesicle protein (semenogelin). Both the gel-bound fibronectin and semenogelin are progressively fragmented and solubilized by the abundant prostatic kallikrein-like protease (prostate-specific antigen) during and after seminal gel liquefaction. Lactoferrin remains essentially unaffected by the seminal proteases.