Maxwell Schubert

The characterization of soluble amyloid prepared in water

Amyloid was extracted from the spleen of a patient with primary amyloidosis by homogenizing it at high speed with water after preliminary treatments, first to remove proteins soluble in saline, and then to remove salts. The extracts containing amyloid appeared to be clear at concentrations up to 6 mg/ml of protein. The material gave little sediment on being centrifuged up to 20,000 g for 1 hr, but the protein was sedimented at 100,000 g in 1 hr. The amyloid could be precipitated from the extracts by addition of NaCl to 0.0075 mole/liter or of CaCl(2) to 0.0025 mole/liter. The protein-bound Congo red formed a red precipitate and this property was used to estimate recovery and purity of amyloid during extraction. On electronmicroscopy the isolated amyloid proved to be morphologically pure. It existed either as single filaments measuring 60-80 A in diameter or as large aggregates of these filaments.Freshly isolated amyloid in water sedimented as a single homogeneous peak with an s degrees (20,[unk]) of about 45-50S. On standing, the solution became cloudy and more rapidly sedimenting components appeared. On electrophoresis the material migrated as a homogeneous peak towards the anode. The protein had an amino acid composition different from that of all known serum proteins. It was rich in acidic amino acids and had little cysteine and methionine and no hydroxyproline. The total content of carbohydrate was less than 2%.

Metachromasia; chemical theory and histochemical use.

\Ieta(’hrommsati(’ stainmimig of tissues was first nn)ti(’e(l in 1875, al)out 20 years tuften’ the i)egisisiinsg of the misanmufacture of synthetic dyes. It was frequently stu(lied thereafter anmd in re(’emit years has been found to i)c of I)an’ti(’ulan’value i nu isist ocheni ical st udies of the commipomiemits of (‘ot i mie(’t I ye t issue. Vet mmueta(’hronuitlti(’ stainsinig is one of the nisore trea(’herolls histochemisical technsi(fues tumid is the sui)ject of frequent and apparently uniresolvai)le (‘ommtroven’sy. Sonmie of the prin(’ipal areas of (‘omitentiomi are the distin(’tioni i)et veenm ‘‘trite” and ‘‘false’’ mmieta(’hmomiiasia, the chenhi(’al miature of the tissue (‘onil)onsetits that stairs mmmeta(‘hn’onmaticallv and evenm the dyes that can undergo a nmieta(’hr()mssati(’ chanige in appropriate tisstles. ()ms the tlseoreti(’al si(le the situation is little i)etter. If there has l)eemi less (‘ommts’oversy over theory tisasi over pra(’ti(’e and immterpretations, it is l)e(’ause fewer theoretical ideas have heenm advamiced anid these have beets ii’idely spa(’ed OVel’ the past half century. Because of considerable recent histodisemisi(’al immtem’est ins mmseta(’hronsmati(’ staimmimig it seenis tinmse to reviemv this field anm(l to try t() I’elate wisat is kimown alxut the (‘henmsistrv of the (lyes, the (‘hronsmotropes, ammd their interactioni ins solution with what is kmiowni about this staimsing process I1I)I)lie(1 to tissues. The purpose of the present review is the rathem’ broad ol of trying to bring togethem’ n’ecesst ideas from several separated fields of study miot always withini the i mmimsmediate s’anige of i midivi(lual workers i nsterested its nssetachronimnut ic stainui nsg. Fun’ insstansce, it is iiot generally appreciate(l that the use of dyes to nmieasun’e tue critical conscenst n’at iotm of nisicelle formmmations of (letergensts is related to nusetach ronmuasia This survey aims to presenit a critical evaluations of numet.hods ani(l ideas that have t’onstrihiuted to use on’ theory n’athem’ thanm to give a (‘onusplele (‘otlu-

The interaction of monosodium urate with connective tissue components

Monosodium urate deposits almost exclusively in the connective tissues of patients with gout. Acetone dried homogenates of bovine nasal cartilage, but not of other tissues, markedly enhances the solubility of urate in buffers having molarities and hydrogen ion concentrations similar to that of most body fluids. The components of cartilage responsible for this effect are the proteinpolysaccharides, compounds of protein and chondroitin sulfate, called PPL. A progressive increase in PPL concentration results in a corresponding increase in urate solubility. If, on the other hand, unbound chondroitin sulfate or PPL digested by trypsin is used, then no significant augmentation of urate solubility occurs indicating that the integrity of the molecule is essential. One subfraction of PPL, PPL(5), causes an even more exaggerated response while another, PPL(3), causes a lesser one. These proteinpolysaccharide macro-molecules also inhibit the crystallization of urate from a supersaturated medium. The mechanism of the solubilizing phenomenon is not known. It is suggested that some type of physical or chemical binding is responsible. When, as a result of normal or accelerated connective tissue turnover, PPL is enzymatically destroyed, urate crystals then precipitate from the saturated tissue fluids.

Improved Method for Determination of Plasma Polysaccharides with Tryptophan.

Summary A method to measure the concentration of sugars in solution, particularly of polysaccharides in biological fluids such as plasma, is presented. It is a modification of a tryptophan method already described and utilizes boric acid in the reaction medium. The color intensity is increased and the color quality modified to give maximum absorption at a longer wave length where interference by non-specific brown produced by reagent blanks and proteins is diminished.

Proteinpolysaccharides from human articular and costal cartilage.

From bovine nasal cartilage two products were isolated, each consisting mainly of chondroitin sulfate and protein (1). These were called proteinpolysaccharides (PP), and the two kinds were distinguished as PP-H and PP-L. Human costal cartilage also yielded two products which, in the extraction and fractionation procedures, behaved rather like the two products from bovine nasal cartilage and were also called PP-H and PP-L (2), although they differed from the corresponding products from bovine cartilage in yield and composition. Isolation and fractionation of the products from human cartilage was far more difficult and gave results more variable than with bovine cartilage. The PP of bovine and human cartilages contain components other than chondroitin sulfate and protein. Partridge and his co-workers (3, 4) found glucosamine and galactose among degradation products of PP from cartilage. Gregory and Roden found keratan sulfate after digestion of bovine nasal PP-L with hyaluronidase and papain (5). Alkaline degradation of bovine nasal PP-L also gave keratan sulfate in an amount 5% of the weight of the PP-L (6). Meyer and his co-workers (7, 8) reported obtaining keratan sulfate from whole human costal cartilage and found this component to increase with age. Anderson (9, 10)

SPECIFIC ADSORPTION OF METACHROMATIC COMPOUNDS OF CHONDROITIN SULFATE BY INSOLUBLE CALCIUM SALTS

A method has been found for specifically adsorbing from solution the metachromatic compounds of chondroitin sulfate and two metachromatic dyes. The specific adsorbants are CaHPO4, CaCO3, and CaC2O4. These adsorb only metachromatic compound and do not adsorb either dye or chromotrope alone. With these adsorbants it is easy to show that the metachromatic compound adsorbed contains dye and chromotrope in equivalent amounts even when the solution from which it was adsorbed contained a tenfold excess of chromotrope. This supports by an independent method the conclusion of an earlier ultracentrifugal study. The evidence presented shows that under the conditions studied there exists in solution a single metachromatic compound of methylene blue and chondroitin sulfate which obeys Beers law over the concentration range 0.37 x 10-4 to 7.2 x 10-4 M, and has a molar extinction coefficient at 570 mµ of 2.36 x 104. Some less specific adsorbants are also mentioned; in addition to adsorbing the metachromatic compound, Ca3(PO4) also adsorbs chromotrope, and Al2O3, dye. The specific adsorbants provide a new tool for the study of metachromasia in solution and facilitate chemical analysis of adsorbable metachromatic compounds.

The proteinpolysaccharides of human costal cartilage

A B S T R A C T Water-soluble proteinpolysaccharides, called PPL, can be extracted from bovine nucleus pulposus in yields of 45%, and from bovine nasal cartilage in yields of 37% of the dry tissue weight. From human costal cartilage only 7% can be extracted. The method used to separate PPL from each of the first two tissues into four distinct fractions separates the PPL of human costal cartilage into four fractions called PPL 3, PPL 4, PPL 5, and PPL 6, which show an increase in protein content, a decrease in chondroitin sulfate content, a nearly constant keratan sulfate content, and an increase in ease of sedimentability and molecular weight. From each of the three tissues mentioned, PPL 3 has a similar amino acid profile and so does PPL 5, but PPL 5 differs from PPL 3 in having a lower content of serine and higher contents of aspartic acid, tyrosine, and arginine. A more extensive effort to characterize these products has been made by analytical ultracentrifugation, and this has led to a further fractionation of PPL 5. Treatment of the cartilage residue or the water-insoluble protein polysaccharide called PPH, with neutral NH20H solution releases water-soluble protein polysaccharides which in composition resemble PPL 4. The water-insoluble residue left after NH2OH treatment, when treated with collagenase, yields two soluble products, one resembling PPL 5 in composition, the other with a much lower chondroitin sulfate and much higher keratan sulfate content. The possibility is suggested that in human costal cartilage, binding of some forms of PPL to collagen may occur.

Proteinpolysaccharide in Connective Tissue: Inhibition of Phase Separation

A macromolecule of protein and chondroitin sulfate (PP-L) inhibits sedimentation of barium-polystyrene sulfonate (BaPSS) and of calcium phosphate at low but not at high values of gravity. Sedimentation of BaPSS removes a large fraction of PP-L from solution, but sedimentation of calcium phosphate does not. The results suggest entanglement among linear polyanionic chains.

Biochemical events in joint disease

FORMERLY untouchable human joints are now receiving the attention of workers in widely separated fields. This is not surprising for there has been a growing realization that in the synovial joint, biochemistry, anatomy and mechanics converge and influence each other in many direct and intimate ways. A movable joint is composed of the synovial membrane and capsule, articular cartilage and subchondral bone, ligaments, tendons and fibrocartilages, and synovial fluid. The most urgent need has been a means to obtain specimens of these joint components from living subjects. This need was perhaps first met by the introduction of safe and effective needles [24] that permitted biopsies of synovial membrane from closed joints of ambulatory patients. Specimens of synovial membrane, cartilage, or bone are now obtained with increasing frequency by orthopedic surgeons during arthrotomy [5, 61, or from amputated limbs [7]. A great deal of information is accumulating from studies on joint components using techniques of chemistry, immunology, histology, cytochemistry, electron microscopy, and immunohistology. Integration and synthesis of this information into a coherent view of joint function is making progress, especially in the normal joint [8, 91. New studies and ideas relate the structure and metabolism of the synovial membrane to its secretion of synovial fluid, and explore the function of this viscous fluid as a nutrient source and lubricant for the moving articular cartilages. This paper will concentrate on some aspects of the altered structure and function of synovial joints involved by rheumatoid arthritis.

Turbidity produced by hexamminecobaltic chloride in serum of rabbits injected intravenously with papain.

Summary 1. Serum of rabbits following injection of chondroitin sulfate, crude papain, or several modifications of papain, became turbid following addition of hexamminecobaltic trivalent cation. Turbidity was directly related to amount of chondroitin sulfate-like material present in the serum. 2. The precipitate produced by this cation contained most of the non-dialysable S35 activity present in the circulation following injection of crude papain, was metachromatic when redissolved, and did not form after incubation of serum with testicular hyaluronidase. 3. Addition of hexamminecobaltic chloride to dilute, dialysed serum is a useful way of demonstrating sulfated mucopolysaccharides in serum at levels exceeding 250 μg/ml.