**2. Identification of the amyloid substance and isolation of its serum precursor**

Amyloids of different clinical settings (also in animals, see **Figure 1**) represent characteristic fibrils under electron microscopy [8]. Therefore, for chemical identification of the amyloid, these fibrils had to be extracted in pure form followed by chromatographic isolation of the major amyloid protein for its chemical analysis by amino acid sequence analysis. The method of isolation of the pure amyloid fibrils was pioneered by Pras et al. [9]. The first amino acid sequence of an amyloid protein was published by Glenner et al. [10], which was derived from a monoclonal immunoglobulin κ-light chain and was named ALκ. The first sequence identifying the chemical nature of inflammation-induced amyloid in monkey and human amyloid was published by Benditt et al. [11], which was named amyloid A (AA). The first anti-AA antibodies were prepared in rabbits where a serum protein in patients suffering from inflammations was detected immunochemically. This protein had an α<sup>1</sup> -electrophoretic mobility and was in serum approximately 180 KDa by calibrated gel filtration [12] and thus ready to monitor the isolation of the soluble with anti-AA reactive precursor. This isolation of serum protein began in summer 1972 and was monitored with another rabbit anti-AA antibody. Its chromatographic separation from serum yielded a native 200 ± 20 kDa AA reactive protein, which was further chromatographically isolated in 5 M guanidine-HCl. The AA reactive protein had an α<sup>2</sup> -electrophoretic mobility and a molecular size of 12.5 kDa. Since this new protein had the same N-terminal amino acid sequence as AA, it was named serum amyloid A (SAA) [13]. Since SAA was larger than AA, a limited proteolytic cleavage had to be presumed in order for the former to generate AA. During the isolation of SAA and its purification to one size by gel filtration, by isoelectric focusing, however, eight SAA bands of different isoelectric point named A-H were identified with anti-AA antibodies (with AAE as the major SAA species for the planned radioimmunoassay), thus indicating the first signs of a polymorphism of SAA [13]. In addition, in plasma, SAA is bound to HDL [14].

**3.2. The molecular size of the SAA and SAA-HDL at different febrile temperatures**

4°C over night as plate B at 4°C followed by room temperature for 6 h similar to plate (a).

Temperature-dependent molecular weight determination of AA-antigenic proteins of acutephase serum (APS) has been performed using an ACA-34 gel filtration column in PBS with the enzyme inhibitor phenylmethylsulfonylfloride (PMSF) under various temperatures as shown in **Figure 3**. The size grading was done by the serum proteins IgM, IgG, albumin and, in addition, cytochrome C and the salt marker N-ε-DNP-lysine. The proteins were identified by way of the size position in the column by immunodiffusion as SAA-HDL at a size of ca. 180–200 kDa or SAA at 12.5 kDa. The different temperatures were kept with a temperature-controlled glass jacket, that is at 37°C in column run A, at 38°C in B, at 40°C in C and at 42°C in D. E was run as D, but without enzyme protection by PMSF, thus showing some degradation of SAA [18].

**Figure 2.** Immunochemical comparison of SAA-HDL, SAA and AA. Immunodiffusion (ID) at different temperatures [14, 15]. The ID was performed in 1.5% Seakem agarose in 0.03 M barbital buffer, pH 8.6 with the same reagents in each of the three plates à 6 wells ((a)-(c)). Top and bottom well contained AA (0.1 mg/ml), the middle well contained polyclonal rabbit anti-AA antibodies undiluted and the 4 side wells contained elevated SAA-HDL containing APS from 4 patients at 1/10 diluted. Plate (a) after diffusion over night at room temperature, plate (b) at 4°C over night and plate (c) first at

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At a normal body temperature of 37°C, AA-containing proteins are at a single position as that of the SAA-HDL stable complex in A (fractions 34–37). However, already at 38°C, the stable complex SAA-HDL begins to dissociate as shown in **Figure 3**, run B. AA antigenic proteins appear at three positions, that is first of all at the void volume at fractions 19–20 (which has not been further analyzed, but could be related to aggregated SAA and/or its derivatives), secondly at the position of the stable SAA-HDL complex at fractions 34–36 and thirdly at the position of the HDL-free SAA at 53–56, as determined by the antigenic differentiation as seen in **Figure 2**. This size differentiation may also indicate functional heterogeneity, as the different affinities of SAA to HDL. This dissociation begins at 38°C and progresses with diminution of the SAA-HDL complex until run C. SAA-HDL disappeared at a "threshold of life" in run D at 42°C and above where the SAA species was maximized and the broadest was seen at fraction 53–56. This shows a temperature-induced gradual dissociation of SAA from HDL at the different febrile temperatures, which was shown here in vitro. This may also occur under systemic and local, acute-phase conditions, with the release of different SAA isotypes at different temperatures, for functions to be discovered. Finally, the SAA monomers released at different temperatures differ
