SummaryTwo-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0 . 5 ns increased to Ϸ 3 . 0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.

and light chain (V L ) variable domains. These two domains associate noncovalently to form the smallest functional antibody protein capable of antigen binding that most closely approximates the Ig molecule (1, 2). These proteins have been previously found to be less stable in terms of domain-domain association than Fab fragments due to the lack of covalent bonds between the two variable domains (3, 4). Single chain antibody (SCA) molecules have been produced to diminish this instability by the introduction of an interdomain linker peptide (5, 6). SCA proteins often mimic the parent antibody active site in terms of antigen binding and structural properties with usually some reduction in affinity for antigen (7-9). Recently, Fv proteins have been engineered to possess an interdomain disulfide linkage, effectively disallowing dissociation of the two domains (10). Due to their small size and amenability to genetic engineering, recombinant Fv proteins have been widely applied in the study of antibody active site structure-function (7, 11-13), idiotypy and metatypy (14 -17), antibody bivalency and bispecificity (18 -21), and in vivo immunodiagnostics and therapy (10,22,23).Fv molecules have been efficacious proteins in the study of antibody active site structure-function and protein stability. Studies involving comparative analysis of Fv protein with other immunoglobulin constructs afford unique opportunities for determining domain-domain interactions and the effects these interactions exert upon the intrinsic conformational and antigen binding properties of the variable domains. Being covalently coupled by an interdomain linker, SCA proteins have been suggested to possess greater interdomain stability than their Fv counterparts due to the favorable entropic effect of domain coupling (6,8). This would, in turn, suggest that in the appropriate Fv molecule (one with high affinity for antigen), interdomain associative properties would dictate the overall affinity displayed for antigen because only associated V L /V H proteins would bind antigen. In previous studies, dissociation constants for the V L /V H association in Fv molecules varied from 10 Ϫ7 to Ͼ10 Ϫ9 M (3, 24 -26). These Fv molecules also displayed similar dissociation constants for their respective antigens (10 Ϫ7 to Ͼ10 Ϫ9 M), further supporting some correlation between interdomain and active site/antigen interactions. Further analysis of V L /V H association constants in relation to antigen affinity would allow identification of components necessary for the production of stable Fv molecules and novel variable domain proteins.Fv molecules have been especially useful in the study of idiotypy and metatypy. Antibody idiotype and metatype are immunologically resolved markers of active site structural and conformational determinants in the unliganded and liganded state, respectively (review in Refs. 27 and 28). Indeed, the transition between the idiotypic and metatypic states upon ligand binding emphasizes the dynamic properties of antibody

Stability measurements of SCA 04-01/212 (anti-ssDNA) which possesses a groove-shaped active site were performed by Gdn-HCl-induced unfolding, analyzed assuming a simple two-state equilibrium, and expressed as the free energy of unfolding, delta Gn-u. A delta Gn-u of 1.44 +/- 0.13 kcal/mol was determined experimentally for SCA 04-01/212. In addition, the conformational stabilities of HCDR transplants, hybrid antibody molecules resulting from the transplantation of HCDRs from SCA 4-4-20 (anti-fluorescein) into the corresponding regions of 04-01 in all combinations, were determined using the identical protocol applied to SCA 04-01. On the basis of the results of these stability experiments, the HCDR transplants were categorized into three groups, representing low, intermediate, and high stability. Data were discussed in terms of the relationships between structure-function and conformational stability pertaining to the groove-shaped antibody active site of SCA 04-01/212 and the pocket-shaped active site of SCA 4-4-20/212.

Oxygen quenching experiments were carried out on zincprotoporphyrin IX reconstituted myoglobin (MbFe+ '") at different temperatures and two solvent viscosities. The data were fit to a dynamic mode1 for quenching of fluorophores in protein interiors previously presented (Biophysical .I., 45 (1984) 789-794). The parameters associated with the oxygen entry rate (k+), exit rate (k-), and migration rate (x> in the protein were obtained at six temperatures and two viscosities (1 and 8 cp), along with the activation enthalpies associated with the above rates (k' and k-1. The partition coefficient (a) was calculated at each temperature along with the free energy, AC", associated with this partition. The rate parameters (kf, k-, x> and the partition coefficient ((Y) have also been determined for the sample in 40% sucrose (8 cp), to evaluate the effect of bulk solvent viscosities on these values. The steady-state Stern-Volmer quenching plot was calculated using the rate parameters obtained from the analysis (of the dynamic model). Comparison of the Stern-Volmer points obtained using the dynamic mode1 and those obtained experimentally showed excellent agreement.